Novel herbicide resistance genes

ABSTRACT

The subject invention provides novel plants that are not only resistant to 2,4-D and other phenoxy auxin herbicides, but also to aryloxyphenoxypropionate herbicides. Heretofore, there was no expectation or suggestion that a plant with both of these advantageous properties could be produced by the introduction of a single gene. The subject invention also includes plants that produce one or more enzymes of the subject invention alone or “stacked” together with another herbicide resistance gene, preferably a glyphosate resistance gene, so as to provide broader and more robust weed control, increased treatment flexibility, and improved herbicide resistance management options. More specifically, preferred enzymes and genes for use according to the subject invention are referred to herein as AAD (aryloxyalkanoate dioxygenase) genes and proteins. No α-ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of different chemical classes and modes of action. This highly novel discovery is the basis of significant herbicide tolerant crop trait opportunities as well as development of selectable marker technology. The subject invention also includes related methods of controlling weeds. The subject invention enables novel combinations of herbicides to be used in new ways. Furthermore, the subject invention provides novel methods of preventing the formation of, and controlling, weeds that are resistant (or naturally more tolerant) to one or more herbicides such as glyphosate.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. Ser. No.17/143,824, filed Jan. 7, 2021, which is a continuation application ofU.S. Ser. No. 15/288,406, filed Oct. 7, 2016, now patented as U.S. Pat.No. 10,947,555, which is a continuation application of U.S. Ser. No.14/820,893, filed Aug. 7, 2015, now patented as U.S. Pat. No.10,174,337, which is a continuation application of U.S. Ser. No.12/951,813, filed Nov. 22, 2010, now patented as U.S. Pat. No.9,127,289, which is a continuation application of U.S. Ser. No.11/587,893, filed May 22, 2008, now patented as U.S. Pat. No. 7,838,733,which is a national stage entry of PCT/US2005/014737, filed May 2, 2005,which claims the benefit of U.S. Provisional Application Ser. No.60/567,052, filed Apr. 30, 2004, which are hereby incorporated byreference in their entirety, including any figures, tables, nucleic acidsequences, amino acid sequences, or drawings.

INCORPORATION OF SEQUENCE LISTING

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herein andidentified as follows: One 42 KB ACII (Text) file named “334171_ST25”created on Apr. 7, 2021.

BACKGROUND OF THE INVENTION

Weeds can quickly deplete soil of valuable nutrients needed by crops andother desirable plants. There are many different types of herbicidespresently used for the control of weeds. One extremely popular herbicideis glyphosate.

Crops, such as corn, soybeans, canola, cotton, sugar beets, wheat, turf,and rice, have been developed that are resistant to glyphosate. Thus,fields with actively growing glyphosate resistant corn, for example, canbe sprayed to control weeds without significantly damaging the cornplants.

With the introduction of genetically engineered, glyphosate tolerantcrops (GTCs) in the mid-1990's, growers were enabled with a simple,convenient, flexible, and inexpensive tool for controlling a widespectrum of broadleaf and grass weeds unparalleled in agriculture.Consequently, producers were quick to adopt GTCs and in many instancesabandon many of the accepted best agronomic practices such as croprotation, herbicide mode of action rotation, tank mixing, incorporationof mechanical with chemical and cultural weed control. Currentlyglyphosate tolerant soybean, cotton, corn, and canola are commerciallyavailable in the United States and elsewhere in the Western Hemisphere.More GTCs (e.g., wheat, rice, sugar beets, turf, etc.) are poised forintroduction pending global market acceptance. Many other glyphosateresistant species are in experimental to development stages (e.g.,alfalfa, sugar cane, sunflower, beets, peas, carrot, cucumber, lettuce,onion, strawberry, tomato, and tobacco; forestry species like poplar andsweetgum; and horticultural species like marigold, petunia, andbegonias; see “isb.vt.edu/cfdocs/fieldtests1.cfm, 2005” website).Additionally, the cost of glyphosate has dropped dramatically in recentyears to the point that few conventional weed control programs caneffectively compete on price and performance with glyphosate GTCsystems.

Glyphosate has been used successfully in burndown and other non-cropareas for total vegetation control for more than 15 years. In manyinstances, as with GTCs, glyphosate has been used 1-3 times per year for3, 5, 10, up to 15 years in a row. These circumstances have led to anover-reliance on glyphosate and GTC technology and have placed a heavyselection pressure on native weed species for plants that are naturallymore tolerant to glyphosate or which have developed a mechanism toresist glyphosate's herbicidal activity.

Extensive use of glyphosate-only weed control programs is resulting inthe selection of glyphosate-resistant weeds, and is selecting for thepropagation of weed species that are inherently more tolerant toglyphosate than most target species (i.e., weed shifts). (Ng et al.,2003; Simarmata et al., 2003; Lorraine-Colwill et al., 2003; Sfiligoj,2004; Miller et al., 2003; Heap, 2005; Murphy et al., 2002; Martin etal., 2002.) Although glyphosate has been widely used globally for morethan 15 years, only a handful of weeds have been reported to havedeveloped resistance to glyphosate (Heap, 2005); however, most of thesehave been identified in the past 3-5 years. Resistant weeds include bothgrass and broadleaf species—Lolium rigidum, Lolium multiflorum, Eleusineindica, Ambrosia artemisiifolia, Conyza canadensis, Conyza bonariensis,and Plantago lanceolata. Additionally, weeds that had previously notbeen an agronomic problem prior to the wide use of GTCs are now becomingmore prevalent and difficult to control in the context of GTCs, whichcomprise >80% of U.S. cotton and soybean acres and >20% of U.S. cornacres (Gianessi, 2005). These weed shifts are occurring predominantlywith (but not exclusively) difficult-to-control broadleaf weeds. Someexamples include Ipomoea, Amaranthus, Chenopodium, Taraxacum, andCommelina species.

In areas where growers are faced with glyphosate resistant weeds or ashift to more difficult-to-control weed species, growers can compensatefor glyphosate's weaknesses by tank mixing or alternating with otherherbicides that will control the missed weeds. One popular andefficacious tankmix partner for controlling broadleaf escapes in manyinstances has been 2,4-diclorophenoxyacetic acid (2,4-D). 2,4-D has beenused agronomically and in non-crop situations for broad spectrum,broadleaf weed control for more than 60 years. Individual cases of moretolerant species have been reported, but 2,4-D remains one of the mostwidely used herbicides globally. A limitation to further use of 2,4-D isthat its selectivity in dicot crops like soybean or cotton is very poor,and hence 2,4-D is not typically used on (and generally not near)sensitive dicot crops. Additionally, 2,4-D's use in grass crops issomewhat limited by the nature of crop injury that can occur. 2,4-D incombination with glyphosate has been used to provide a more robustburndown treatment prior to planting no-till soybeans and cotton;however, due to these dicot species' sensitivity to 2,4-D, theseburndown treatments must occur at least 14-30 days prior to planting(Agriliance, 2003).

2,4-D is in the phenoxy acid class of herbicides, as are MCPA, mecoprop,and dichlorprop. 2,4-D has been used in many monocot crops (such ascorn, wheat, and rice) for the selective control of broadleaf weedswithout severely damaging the desired crop plants. 2,4-D is a syntheticauxin derivative that acts to deregulate normal cell-hormone homeostasisand impede balanced, controlled growth; however, the exact mode ofaction is still not known.

2,4-D has different levels of selectivity on certain plants (e.g.,dicots are more sensitive than grasses). Differential metabolism of2,4-D by different plants is one explanation for varying levels ofselectivity. In general, plants metabolize 2,4-D slowly, so varyingplant response to 2,4-D may be more likely explained by differentactivity at the target site(s) (WSSA, 2002). Plant metabolism of 2,4-Dtypically occurs via a two-phase mechanism, typically hydroxylationfollowed by conjugation with amino acids or glucose (WSSA, 2002).

Over time, microbial populations have developed an alternative andefficient pathway for degradation of this particular xenobiotic, whichresults in the complete mineralization of 2,4-D. Successive applicationsof the herbicide select for microbes that can utilize the herbicide as acarbon source for growth, giving them a competitive advantage in thesoil. For this reason, 2,4-D is currently formulated to have arelatively short soil half-life, and no significant carryover effects tosubsequent crops are encountered. This adds to the herbicidal utility of2,4-D.

One organism that has been extensively researched for its ability todegrade 2,4-D is Ralstonia eutropha (Streber et al., 1987). The genethat codes for the first enzymatic step in the mineralization pathway istfdA. See U.S. Pat. No. 6,153,401 and GENBANK Acc. No. M16730. TfdAcatalyzes the conversion of 2,4-D acid to dichlorophenol (DCP) via anα-ketoglutarate-dependent dioxygenase reaction (Smejkal et al., 2001).DCP has little herbicidal activity compared to 2,4-D. TfdA has been usedin transgenic plants to impart 2,4-D resistance in dicot plants (e.g.,cotton and tobacco) normally sensitive to 2,4-D (Streber et al. (1989),Lyon et al. (1989), Lyon (1993), and U.S. Pat. No. 5,608,147).

A large number of tfdA-type genes that encode proteins capable ofdegrading 2,4-D have been identified from the environment and depositedinto the Genbank database. Many homologues are similar to tfdA (>85%amino acid identity) and have similar enzymatic properties to tfdA.However, there are a number of homologues that have a significantlylower identity to tfdA (25-50%), yet have the characteristic residuesassociated with α-ketoglutarate dioxygenase Fe⁺² dioxygenases. It istherefore not obvious what the substrate specificities of thesedivergent dioxygenases are.

One unique example with low homology to tfdA (28% amino acid identity)is rdpA from Sphingobium herbicidovorans (Kohler et al., 1999,Westendorf et al., 2002). This enzyme has been shown to catalyze thefirst step in (R)-dichlorprop (and other (R)-phenoxypropionic acids) aswell as 2,4-D (a phenoxyacetic acid) mineralization (Westendorf et al.,2003). Although the organisms that degrade phenoxypropionic acid weredescribed some time ago, little progress had been made in characterizingthis pathway until recently (Horvath et al., 1990). An additionalcomplication to dichlorprop degradation is the stereospecificity (R vs.S) involved in both the uptake (Kohler, 1999) and initial oxidation ofdichlorprop (Westendorf et al., 2003). Heterologous expression of rdpAin other microbes, or transformation of this gene into plants, has notheretofore been reported. Literature has focused primarily around closehomologues of tfdA that primarily degrade achiral phenoxyacetic acids(e.g., 2,4-D).

Development of new herbicide-tolerant crop (HTC) technologies has beenlimited in success due largely to the efficacy, low cost, andconvenience of GTCs. Consequently, a very high rate of adoption for GTCshas occurred among producers. This created little incentive fordeveloping new HTC technologies.

Aryloxyalkanoate chemical substructures are a common entity of manycommercialized herbicides including the phenoxy auxins (such as 2,4-Dand dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr),aryloxyphenoxypropionates (AOPP) acetyl-coenzyme A carboxylase (ACCase)inhibitors (such as haloxyfop, quizalofop, and diclofop), and5-substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors(such as pyraflufen and flumiclorac). However, these classes ofherbicides are all quite distinct, and no evidence exists in the currentliterature for common degradation pathways among these chemical classes.Discovery of a multifunctional enzyme for the degradation of herbicidescovering multiple modes would be both unique and valuable as an HTCtrait.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides novel plants that are not only resistantto 2,4-D, but also to AOPP herbicides. Heretofore, there was noexpectation or suggestion that a plant with both of these advantageousproperties could be produced by the introduction of a single gene. Thesubject invention also includes plants that produce one or more enzymesof the subject invention “stacked” together with one or more otherherbicide resistance genes, including, but not limited to, glyphosate-,imidazolinone-, and glufosinate-resistance genes, so as to provideherbicide-tolerant plants compatible with broader and more robust weedcontrol and herbicide resistance management options. The presentinvention further includes methods and compositions utilizing homologuesof the genes and proteins exemplified herein.

In some embodiments, the invention provides monocot and dicot plantstolerant to 2,4-D, AOPP, and one or more commercially availableherbicides (e.g., glyphosate, imidazolinones, glufosinate,sulfonylureas, dicamba, bromoxynil, and others). Vectors comprisingnucleic acid sequences responsible for such herbicide tolerance are alsodisclosed, as are methods of using such tolerant plants and combinationsof herbicides for weed control and prevention of weed population shifts.The subject invention enables novel combinations of herbicides to beused in new ways. Furthermore, the subject invention provides novelmethods of preventing the development of, and controlling, strains ofweeds that are resistant to one or more herbicides such as glyphosate.The subject invention enables novel uses of novel combinations ofherbicides and crops, including preplant application to an area to beplanted immediately prior to planting with seed for plants that wouldotherwise be sensitive to that herbicide (such as 2,4-D).

The subject invention relates in part to the identification of an enzymethat is not only able to degrade 2,4-D, but also surprisingly possessesnovel properties, which distinguish the enzyme of the subject inventionfrom previously known tfdA proteins, for example. More specifically, thesubject invention relates to the use of an enzyme that is capable ofdegrading both 2,4-D and AOPP herbicides, in an enantiospecific manner.No α-ketoglutarate-dependent dioxygenase enzyme has previously beenreported to have the ability to degrade herbicides of different chemicalclasses and modes of action. The preferred enzyme and gene for useaccording to the subject invention are referred to herein as AAD-1(AryloxyAlkanoate Dioxygenase). This highly novel discovery is the basisof significant HTC trait and selectable marker opportunities.

There was no prior motivation to produce plants comprising an AAD-1 gene(preferably an AAD-1 polynucleotide that has a sequence optimized forexpression in one or more types of plants, as exemplified herein), andthere was no expectation that such plants could effectively produce anAAD-1 enzyme to render the plants resistant to not only phenoxy acidherbicides (such as 2,4-D) but also AOPP herbicides (such as quizalofop,haloxyfop, et al.). Thus, the subject invention provides many advantagesthat were not heretofore thought to be possible in the art.

This invention also relates in part to the identification and use ofgenes encoding aryloxyalkanoate dioxygenase enzymes that are capable ofdegrading phenoxy auxin and aryloxyphenoxypropionate herbicides. Methodsof screening proteins for these activities are within the scope of thesubject invention. Thus, the subject invention includes degradation of2,4-dichlorophenoxyacetic acid, other phenoxyalkanoate auxin herbicides,and aryloxyphenoxypropionate herbicides by a recombinantly expressedAAD-1 enzyme. The subject invention also includes methods of controllingweeds wherein said methods comprise applying one or more AOPP, phenoxyauxin, or other aryloxyalkanoate herbicides to plants comprising anAAD-1 gene. The subject invention also provides methods of using anAAD-1 gene as a selectable marker for identifying plant cells and wholeplants transformed with AAD-1, optionally including one, two, or moreexogenous genes simultaneously inserted into target plant cells. Methodsof the subject invention include selecting transformed cells that areresistant to appropriate levels of an herbicide. The subject inventionfurther includes methods of preparing a polypeptide, having thebiological activity of aryloxyalkanoate dioxygenase, by culturing plantsand/or cells of the subject invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general scheme for dioxygenase cleavage of phenoxy auxinor AOPP herbicides.

FIG. 2 shows loss of herbicidal activity from a 2,4-D solution treatedwith AAD-1.

FIG. 3 shows loss of herbicidal activity from a haloxyfop solutiontreated with AAD-1.

FIG. 4 shows anticipated phenols produced from representative herbicidescatalyzed by AAD-1.

FIG. 5 shows 2,4-dichlorophenol production by recombinant AAD-1.

FIGS. 6A and 6B show phenol production by recombinant AAD-1 from variousherbicide substrates.

FIG. 7 shows AAD-1 reaction rate to substrate concentration for fourherbicide substrates.

FIGS. 8A and 8B show that AAD-1 (v3) was expressed equally inArabidopsis leaves of different ages but continued to accumulatethroughout the 25 days of experiment. Plants that were not sprayed withthe herbicide 2,4-D (panel A) expressed a little more AAD-1 (v3) thanthose had been sprayed (panel B). Bars represent the mean±SEM of 5leaves from 5 different plants, with percent expression of AAD-1 (v3)normalized to total soluble protein. Light bars represent the thirdyoung leaves (N−3) collected from the top, dark bars represent the 5tholdest leaves from the bottom.

FIGS. 9A, 9B, and 9C show injury of Arabidopsis plants after 2,4-Dtreatment. Four different lines were each treated with four differentdoses of 2,4-D and their injury was graded 4 (panel A) and 14 (panel B)days after treatment. Their expression of AAD-1 (v3) in leaves was alsodetermined using ELISA (panel C). The results were mean±SEM of fiveleaves from five different plants received the same treatment.

FIG. 10 illustrates that pDAB3230-transformed Arabidopsis (AAD-1+EPSPS)shows >14-fold level of glyphosate tolerance 7 DAT vs. wildtype andtransformed control Arabidopsis lines.

FIG. 11 shows dose response of callused maize suspensions toR-haloxyfop.

FIG. 12 shows that at 1 μM cyhalofop phenol, growth is still 76% as highas the control without cyhalofop phenol.

FIG. 13 illustrates dose-response data on one transgenic event,3404-006, to haloxyfop.

FIG. 14 shows the responses of several AAD-1 (v3)-transformed andnon-transformed event clones to lethal doses of two AOPP herbicides(haloxyfop and quizalofop) applied as a postemergence spray 1 weekprior.

FIG. 15 shows three different T2 lineages from 3404 transformations thatwere pre-screened with Liberty® to remove nulls, which were chosen tocompare their tolerance to quizalofop with respect to their AAD-1expression. Expression was measured at 14 DAT (data not shown) and at 30DAT.

FIG. 16 shows AAD-1 (v3)-transformed corn tolerant to 8× field rates ofquizalofop (Assure II) under field conditions.

FIG. 17 illustrates data from immature maize embryos grown oncyhalofop-containing media.

FIG. 18 shows Western Blotting analysis on soybean calli transformedwith AAD-1 (V3) gene indicating that the callus cells are expressingAAD-1 (v3) protein.

FIG. 19 shows fitted curves for 2,4-D degradation rates by AAD-2 (v1)vs. AAD-1 (v1).

FIG. 20 shows the response of AAD-1 v3 (plant optimized), or AAD-1 (v2)(native), AAD-2 (v1) (native), or AAD-2 (v2) (plantoptimized)-transformed T₁ Arabidopsis to a range of 2,4-D rates appliedpostemergence. Each pot represents an individual transformation eventwithin each gene T₁ family.

FIG. 21 shows western blot analysis of individual native AAD-2(v1)-transformed T₁ Arabidopsis plants. This shows that plantsexpressing the AAD-2 (v1) protein are suffering severe injury from 200 gae (acid equivalent)/ha 2,4-D treatments, which normally causes littleinjury to native AAD-1 (v2) or plant optimized AAD-1 (v3)-transformedArabidopsis. AAD-2 protein is identified on the gel. Several backgroundbands were detected in AAD-2-transformed and Pat/C/F-transformedsamples.

FIG. 22 shows that the relative AAD-2 (v1) activity on the substrateswas 2,4-D=dichlorprop>(R,S)-haloxyfop>>(R)-haloxyfop.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the sequence of a forward primer used to amplify therdpA/AAD-1 (v1) gene.

SEQ ID NO:2 is the sequence of a reverse primer used to amplify therdpA/AAD-1 (v1) gene.

SEQ ID NO:3 is the nucleotide sequence of AAD-1 (v1) from Sphingobiumherbicidovorans.

SEQ ID NO:4 is the nucleic acid sequence of the native AAD-1 gene withinternal NotI restriction site removed. This gene is designated AAD-1(v2). DNA sequencing confirmed that the correct PCR product wasgenerated, but an inadvertent change was made at amino acid #212 fromarginine to cysteine.

SEQ ID NO:5 is a “plant-optimized” DNA sequence AAD-1 (v3). This “gene”encodes SEQ ID NO:11, which is the same as SEQ ID NO:9 except for theaddition of an alanine residue at the second position. The additionalalanine codon (GCT) was included to encode an Nco I site (CCATGG)spanning the ATG start codon, to enable subsequent cloning operations.

SEQ ID NO:6 (“rdpA(ncoI)”) and SEQ ID NO:7 (“3′ saci”) were used toamplify a DNA fragment using the Fail Safe PCR System (Epicenter).

SEQ ID NO:8 is another PCR primer (“BstEII/Del NotI”) that was used withthe “3′ SacI” primer.

SEQ ID NO:9 is the native amino acid sequence encoded by the AAD-1 (v1)gene from Sphingobium herbicidovorans.

SEQ ID NO:10 is the amino acid sequence encoded by the AAD-1 (v2) DNAsequence of SEQ ID NO:4.

SEQ ID NO:11 is the amino acid sequence encoded by the AAD-1 (v3)plant-optimized DNA sequence of SEQ ID NO:5.

SEQ ID NO:12 is the DNA sequence of the native AAD-2 (v1) gene.

SEQ ID NO:13 is the amino acid sequence of the AAD-2 (v1) protein.

SEQ ID NO:14 is a forward primer used to amplify AAD-2 (v1) DNA forcloning.

SEQ ID NO:15 is a reverse primer used to amplify AAD-2 (v1) DNA forcloning.

SEQ ID NO:16 is the M13 forward primer.

SEQ ID NO:17 is the M13 reverse primer.

SEQ ID NO:18 is a forward primer used to amplify AAD-2 (v1) DNA forcloning.

SEQ ID NO:19 is a reverse primer used to amplify AAD-2 (v1) DNA forcloning.

SEQ ID NO:20 is the native soybean EPSPS protein.

SEQ ID NO:21 is a doubly mutated soybean EPSPS protein sequence,containing a mutation at residue 183 (threonine of native proteinreplaced with isoleucine), and at residue 187 (proline in native proteinreplaced with serine).

SEQ ID NO:22 is the soybean-biased DNA sequence that encodes the EPSPSprotein of SEQ ID NO:21.

SEQ ID NO:23 is primer Pat 5-3.

SEQ ID NO:24 is primer Pat 3-3.

SEQ ID NO:25 is forward primer AAD-1 PTU.

SEQ ID NO:26 is reverse primer AAD-1 PTU.

SEQ ID NO:27 is the forward primer for the Coding Region PCR AAD-1.

SEQ ID NO:28 is the reverse primer for the Coding Region PCR AAD-1.

SEQ ID NO:29 is the AAD-2 (v2) nucleotide (plant optimized).

SEQ ID NO:30 is the translated AAD-2 (v2) protein sequence.

SEQ ID NO:31 is the Southern fragment PCR AAD-1 forward primer.

SEQ ID NO:32 is the Southern fragment PCR AAD-1 reverse primer.

DETAILED DESCRIPTION OF THE INVENTION

The subject development of a 2,4-D resistance gene and subsequentresistant crops provides excellent options for controlling broadleaf,glyphosate-resistant (or highly tolerant and shifted) weed species forin-crop applications. 2,4-D is a broad-spectrum, relatively inexpensive,and robust broadleaf herbicide that would provide excellent utility forgrowers if greater crop tolerance could be provided in dicot and monocotcrops alike. 2,4-D-tolerant transgenic dicot crops would also havegreater flexibility in the timing and rate of application. An additionalutility of an herbicide tolerance trait for 2,4-D would be its utilityto prevent damage to normally sensitive crops from 2,4-D drift,volatilization, inversion (or other off-site movement phenomenon),misapplication, vandalism and the like. An additional benefit of theAAD-1 gene is that unlike all tfdA homologues characterized to date,AAD-1 is able to degrade the R-enantiomers (herbicidally active isomers)of the chiral phenoxy auxins (e.g., dichlorprop and mecoprop) inaddition to achiral phenoxy auxins (e.g., 2,4-D, MCPA,4-chlorophenoxyacetic acid). See Table 1. Multiple mixes of differentphenoxy auxin combinations have been used globally to address specificweed spectra and environmental conditions in various regions. Use of theAAD-1 gene in plants would afford protection to a much wider spectrum ofphenoxy auxin herbicides, thereby increasing the flexibility and spectraof weeds that can be controlled, protecting from drift or other off-sitephenoxy herbicide injury for the full breadth of commercially availablephenoxy auxins.

Table 1. Commercially available phenoxy auxins. Reference to phenoxyauxin herbicides is generally made to the active acid but some arecommercially formulated as any of a variety of corresponding esterformulations and these are likewise considered as substrates for AAD-1enzyme in planta as general plant esterases convert these esters to theactive acids in planta. Likewise reference can also be for thecorresponding organic or inorganic salt of the corresponding acid. Whenchiral propionic acid, salt, or ester herbicides are indicated, racemic(R,S) or optically purified (R or S) enantiomers are considered the sameherbicides for the purpose of naming these herbicides, even thoughdifferent CAS numbers may correspond to optically pure compounds.Possible use rate ranges can be as stand-alone treatments or incombination with other herbicides in both crop and non-crop uses.

TABLE 1 Commercially available phenoxy auxins. Preferred Possible useuse rate Chemical rate ranges ranges name CAS no (g ae/ha) (g ae/ha)Structure 2,4-D  94-75-7 25-4000 280-1120

2,4,5-T  93-76-5 25-4000  25-4000

4-CPA  122-88-3 25-4000  25-4000

3,4-DA  588-22-7 25-4000  25-4000

MCPA  94-74-6 25-4000 125-1550

Dichlorprop  120-36-5 25-12000 100-2240

Mecoprop 7085-19-0 25-4000 250-3360

Cloprop  101-10-0 25-4000  25-4000

4-CPP 3307-39-9 25-4000  25-4000

Fenoprop  93-72-1 25-4000  25-4000

3,4-DP 3307-41-3 25-4000  25-4000

An additional benefit of the AAD-1 gene is its unprecedented ability toconcomitantly degrade a host of commercial and non-commercialgraminicidal compounds of the general class aryloxyphenoxypropionates(AOPPs). See Table 2. This attribute may allow the use of any of anumber of AOPP compounds in transgenic crops containing AAD-1, wheretolerance in those crops had not previously warranted use in thosecrops. These will most commonly include grass crops such as corn, rice,wheat, barley, rye, oats, sorghum, warm and cool-season turf species,grass pasture species, and many others, but could also include dicotcrops where AOPP tolerance (naturally present in most dicots) is not atcommercially acceptable levels to allow AOPP use in said dicot crop.

Table 2. AOPP graminicidal compounds listed by accepted common namesReference to AOPP herbicides is generally made to the active acid butmost are commercially formulated as any of a variety of correspondingester formulations and these are likewise considered as substrates forAAD-1 enzyme in planta as general plant esterases convert these estersto the active acids in planta. Likewise reference can also be for thecorresponding organic or inorganic salt of the corresponding acid. Whenchiral propionic acid, salt, or ester herbicides are indicated, racemic(R,S) or optically purified (R or S) enantiomers are considered the sameherbicides for the purpose of naming these herbicides, even thoughdifferent CAS numbers may correspond to optically pure compounds.Possible use rate ranges can be as stand-alone treatments or incombination with other herbicides in both crop and non-crop uses.

TABLE 2 AOPP graminicidal compounds listed by accepted common namesPossible Preferred use rate use rate Chemical ranges ranges name CAS no(g ae/ha) (g ae/ha) Structure Chlorazifop 72492-94-7 10-2000 10-2000

Clodinafop 105512-06-9 10-2000 20-200

Clofop 59621-49-7 10-2000 10-2000

Cyhalofop 122008-85-9 10-2000 105-560

Diclofop 71283-65-3 10-2000 280-2000

Fenoxaprop 66441-23-4 10-2000 20-200

Fenthiaprop 95721-12-3 10-2000 10-2000

Fluazifop 69335-91-7 10-2000 25-420

Haloxyfop 69806-40-2 10-2000 20-600

Isoxapyrifop 87757-18-4 10-2000 30-240

Metamifop 256412-89-2 10-2000 35-280

Propaquizafop 111479-05-1 10-2000 30-240

Quizalofop 76578-14-8 10-2000 20-240

Trifop 58597-74-4 10-2000 10-2000

A single gene (AAD-1) has now been identified which, when geneticallyengineered for expression in plants, has the properties to allow the useof phenoxy auxin herbicides in plants where inherent tolerance neverexisted or was not sufficiently high to allow use of these herbicides.Additionally, AAD-1 can provide protection in planta to AOPP herbicideswhere natural tolerance also was not sufficient to allow selectivity.Plants containing AAD-1 alone now may be treated sequentially or tankmixed with one, two, or a combination of several phenoxy auxinherbicides. The rate for each phenoxy auxin herbicide may range from 25to 4000 g ae/ha, and more typically from 100 to 2000 g ae/ha for thecontrol of a broad spectrum of dicot weeds. Likewise, one, two, or amixture of several AOPP graminicidal compounds may be applied to plantsexpressing AAD-1 with reduced risk of injury from said herbicides. Therate for each AOPP may range from 10 to 2000 g ae/ha, and more typicallyfrom 20-500 g ae/ha for the control of a broad spectrum of monocotweeds. Combinations of these different chemistry classes and herbicideswith different modes of action and spectra in the same field (eithersequentially or in tank mix combination) shall provide control of mostpotential weeds for which herbicidal control is desired.

Glyphosate is used extensively because it controls a very wide spectrumof broadleaf and grass weed species. However, repeated use of glyphosatein GTCs and in non-crop applications has, and will continue to, selectfor weed shifts to naturally more tolerant species orglyphosate-resistant biotypes. Tankmix herbicide partners used atefficacious rates that offer control of the same species but havingdifferent modes of action is prescribed by most herbicide resistancemanagement strategies as a method to delay the appearance of resistantweeds. Stacking AAD-1 with a glyphosate tolerance trait (and/or withother herbicide-tolerance traits) could provide a mechanism to allow forthe control of glyphosate resistant weed species (either grass weedspecies with one or more AOPP herbicides, or broadleaf weed species withone or more phenoxy auxins) in GTCs by enabling the use of glyphosate,phenoxy auxin(s) (e.g., 2,4-D) and AOPP herbicide(s) (e.g., quizalofop)selectively in the same crop. Applications of these herbicides could besimultaneously in a tank mixture comprising two or more herbicides ofdifferent modes of action; individual applications of single herbicidecomposition in sequential applications as pre-plant, preemergence, orpostemergence and split timing of applications ranging from 2 hours to 3months; or, alternatively, any combination of any number of herbicidesrepresenting each chemical class can be applied at any timing within 7months of planting the crop up to harvest of the crop (or the preharvestinterval for the individual herbicide, whichever is shortest).

It is important to have flexibility in controlling a broad spectrum ofgrass and broadleaf weeds in terms of timing of application, rate ofindividual herbicides, and the ability to control difficult or resistantweeds. Glyphosate applications in a crop with a glyphosate resistancegene/AAD-1 stack could range from 250-2500 g ae/ha; phenoxy auxinherbicide(s) (one or more) could be applied from 25-4000 g ae/ha; andAOPP herbicide(s) (one or more) could be applied from 10-2000 g ae/ha.The optimal combination(s) and timing of these application(s) willdepend on the particular situation, species, and environment, and willbe best determined by a person skilled in the art of weed control andhaving the benefit of the subject disclosure.

Herbicide formulations (e.g., ester, acid, or salt formulation; orsoluble concentrate, emulsifiable concentrate, or soluble liquid) andtankmix additives (e.g., adjuvants or compatibility agents) cansignificantly affect weed control from a given herbicide or combinationof one or more herbicides. Any combination of these with any of theaforementioned herbicide chemistries is within the scope of thisinvention.

One skilled in the art would also see the benefit of combining two ormore modes of action for increasing the spectrum of weeds controlledand/or for the control of naturally more tolerant species or resistantweed species could also extend to chemistries for which herbicidetolerance was enabled in crops through human involvement (eithertransgenically or non-transgenically) beyond GTCs. Indeed, traitsencoding glyphosate resistance (e.g., resistant plant or bacterialEPSPS, GOX, GAT), glufosinate resistance (e.g., Pat, bar), acetolactatesynthase (ALS)-inhibiting herbicide resistance (e.g., imidazolinone,sulfonylurea, triazolopyrimidine sulfonanilide, pyrmidinylthiobenzoates,and other chemistries=AHAS, CsrI, SurA, et al.), bromoxynil resistance(e.g., Bxn), resistance to inhibitors of HPPD(4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitorsof phytoene desaturase (PDS), resistance to photosystem II inhibitingherbicides (e.g., psbA), resistance to photosystem I inhibitingherbicides, resistance to protoporphyrinogen oxidase IX (PPO)-inhibitingherbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g.,CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), andothers could be stacked alone or in multiple combinations to provide theability to effectively control or prevent weed shifts and/or resistanceto any herbicide of the aforementioned classes.

Regarding additional herbicides, some additional preferred ALSinhibitors include the triazolopyrimidine sulfonanilides (such ascloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, andpenoxsulam), pyrimidinylthiobenzoates (such as bispyribac andpyrithiobac), and flucarbazone. Some preferred HPPD inhibitors includemesotrione, isoxaflutole, and sulcotrione. Some preferred PPO inhibitorsinclude flumiclorac, flumioxazin, flufenpyr, pyraflufen, fluthiacet,butafenacil, carfentrazone, sulfentrazone, and the diphenylethers (suchas acifluorfen, fomesafen, lactofen, and oxyfluorfen).

Additionally, AAD-1 alone or stacked with one or more additional HTCtraits can be stacked with one or more additional input (e.g., insectresistance, fungal resistance, or stress tolerance, et al.) or output(e.g., increased yield, improved oil profile, improved fiber quality, etal.) traits. Thus, the subject invention can be used to provide acomplete agronomic package of improved crop quality with the ability toflexibly and cost effectively control any number of agronomic pests.

The subject invention relates in part to the identification of an enzymethat is not only able to degrade 2,4-D, but also surprisingly possessesnovel properties, which distinguish the enzyme of the subject inventionfrom previously known tfdA proteins, for example. Even though thisenzyme has very low homology to tfdA, the genes of the subject inventioncan still be generally classified in the same overall family ofα-ketoglutarate-dependent dioxygenases. This family of proteins ischaracterized by three conserved histidine residues in a“HX(D/E)X₂₃₋₂₆(T/S)X₁₁₄₋₁₈₃HX₁₀₋₁₃R” motif (SEQ ID NO: 33) whichcomprises the active site. The histidines coordinate Fe²⁺ ion in theactive site that is essential for catalytic activity (Hogan et al.,2000). The preliminary in vitro expression experiments discussed hereinwere tailored to help select for novel attributes.

More specifically, the subject invention relates in part to the use ofan enzyme that is not only capable of degrading 2,4-D, but also AOPPherbicides. No α-ketoglutarate-dependent dioxygenase enzyme haspreviously been reported to have the ability to degrade herbicides ofdifferent chemical classes and modes of action. Preferred enzymes andgenes for use according to the subject invention are referred to hereinas AAD-1 (AryloxyAlkanoate Dioxygenase) genes and proteins. As disclosedherein a peptide of SEQ ID NO: 9 is provided that is capable ofdegrading 2,4-D, and AOPP herbicides. Mapping theα-ketoglutarate-dependent dioxygenase motif disclosed in the previousparagraph to the sequence of SEQ ID NO: 9 reveals the AAD-1 motif ofHX₁₁₂D(X)₁₁₄₋₁₃₇T(X)₁₃₉₋₂₆₉H(X)₂₇₁₋₂₈₀R (SEQ ID NO: 34), wherein

X₁₁₂ represents a single amino acid at position 112, relative to thesequence of SEQ ID NO: 9;

(X)₁₁₄₋₁₃₇ represents a sequence of 24 amino acids; (X)₁₃₉₋₂₆₉represents a sequence of 131 amino acids; and

-   -   (X)₂₇₁₋₂₈₀ represents a sequence of 10 amino acids.

This invention also relates in part to the identification and use ofgenes encoding aryloxyalkanoate dioxygenase enzymes that are capable ofdegrading phenoxy auxin and aryloxyphenoxypropionate herbicides. Thus,the subject invention relates in part to the degradation of2,4-dichlorophenoxyacetic acid, other phenoxyalkanoic auxin herbicides,and aryloxyphenoxyalkanoate herbicides by a recombinantly expressedAAD-1 enzyme.

The subject proteins tested positive for 2,4-D conversion to2,4-dichlorophenol (“DCP”; herbicidally inactive) in analytical andbiological assays. Partially purified proteins of the subject inventioncan rapidly convert 2,4-D to DCP (ranging from 50-100% conversion) invitro. An additional advantage that AAD-1-transformed plants provide isthat parent herbicide(s) are metabolized to inactive forms, therebyreducing the potential for harvesting herbicidal residues in grain orstover.

The subject invention also includes methods of controlling weeds whereinsaid methods comprise applying an AOPP herbicide and/or a phenoxy auxinherbicide to plants comprising an AAD-1 gene.

In light of these discoveries, novel plants that comprise apolynucleotide encoding this type of enzyme are now provided.Heretofore, there was no motivation to produce such plants, and therewas no expectation that such plants could effectively produce thisenzyme to render the plants resistant to not only phenoxy acidherbicides (such as 2,4-D) but also AOPP herbicides. Thus, the subjectinvention provides many advantages that were not heretofore thought tobe possible in the art.

Publicly available strains (deposited in culture collections like ATCCor DSMZ) can be acquired and screened, using techniques disclosedherein, for novel genes. Sequences disclosed herein can be used toamplify and clone the homologous genes into a recombinant expressionsystem for further screening and testing according to the subjectinvention.

As discussed above in the Background section, one organism that has beenextensively researched for its ability to degrade 2,4-D is Ralstoniaeutropha (Streber et al., 1987). The gene that codes for the firstenzyme in the degradation pathway is tfdA. See U.S. Pat. No. 6,153,401and GENBANK Acc. No. M16730. TfdA catalyzes the conversion of 2,4-D acidto herbicidally inactive DCP via an α-ketoglutarate-dependentdioxygenase reaction (Smejkal et al., 2001). TfdA has been used intransgenic plants to impart 2,4-D resistance in dicot plants (e.g.,cotton and tobacco) normally sensitive to 2,4-D (Streber et al., 1989;Lyon et al., 1989; Lyon et al., 1993). A large number of tfdA-type genesthat encode proteins capable of degrading 2,4-D have been identifiedfrom the environment and deposited into the Genbank database. Manyhomologues are quite similar to tfdA (>85% amino acid identity) and havesimilar enzymatic properties to tfdA. However, a small collection ofα-ketoglutarate-dependent dioxygenase homologues are presentlyidentified that have a low level of homology to tfdA.

RdpA, from Sphingobium herbicidovorans (Westendorf et al., 2002), is oneunique example with low homology (28% amino acid identity). This enzymehas been shown to catalyze the first step in (R)-dichlorprop (and other(R)-phenoxypropionic acids) as well as 2,4-D (a phenoxyacetic acid)mineralization (Westendorf et al., 2003). Although the organismresponsible for phenoxypropionic acid degradation has been known forsome time, little progress has been made in characterizing this pathwayuntil recently (Horvath et al., 1990). An additional complication todichlorprop degradation is the stereospecificity (R vs. S) involved inboth the uptake (Kohler, 1999) and initial oxidation of dichlorprop(Westendorf et al., 2003). Heterologous expression of rdpA in othermicrobes or transformation of this gene into plants, heretofore, was notreported. Literature has focused primarily around close homologues oftfdA that primarily degrade achiral phenoxyacetic acids. There was noprior expectation that rdpA or AAD-1 genes could be successfullyexpressed in plants to render the plants resistant to 2,4-D (not tomention the completely surprising AOPP resistance).

As described in more detail in the Examples below, rdpA was cloned fromSphingobium herbicidovorans and tested for substrate promiscuity amongvarious herbicide chemical classes. This α-ketoglutarate-dependentdioxygenase enzyme purified in its native form had previously been shownto degrade 2,4-D and dichlorprop (Westendorf et al., 2002 and 2003).However, no α-ketoglutarate-dependent dioxygenase enzyme has previouslybeen reported to have the ability to degrade herbicides of differentchemical classes and modes of action. RdpA has never been expressed inplants, nor was there any motivation to do so because development of newHTC technologies has been limited due largely to the efficacy, low cost,and convenience of GTCs (Devine, 2005).

In light of the novel activity, proteins and genes of the subjectinvention are referred to herein as AAD-1 proteins and genes. AAD-1 waspresently confirmed to degrade a variety of phenoxyacetic andphenoxypropionic auxin herbicides in vitro. However, this enzyme, asreported for the first time herein, was surprisingly found to also becapable of degrading additional substrates of the class ofaryloxyalkanoate molecules. Substrates of significant agronomicimportance are the aryloxyphenoxypropionate (AOPP) grass herbicides.This highly novel discovery is the basis of significant HerbicideTolerant Crop (HTC) and selectable marker trait opportunities.

The broad spectrum grass AOPP herbicides are reported herein to beexcellent substrates for AAD-1 as well as 2,4-D, dichlorprop, and otherphenoxy auxins. This enzyme is unique in its ability to deliverherbicide degradative activity to a range of broad spectrum broadleafherbicides (phenoxy auxins) and a range of broad spectrum, highly activegrass herbicides (AOPPs).

Thus, the subject invention relates in part to the degradation of2,4-dichlorophenoxyacetic acid, other phenoxyalkanoic auxin herbicides,and aryloxyphenoxy-alkanoate herbicides by a recombinantly expressedaryloxyalkanoate dioxygenase enzyme (AAD-1). This invention also relatesin part to identification and uses of genes encoding an aryloxyalkanoatedioxygenase degrading enzyme (AAD-1) capable of degrading phenoxy auxinand aryloxyphenoxypropionate herbicides.

The subject enzyme enables transgenic expression resulting in toleranceto combinations of herbicides that would control nearly all broadleafand grass weeds. AAD-1 can serve as an excellent herbicide tolerant crop(HTC) trait to stack with other HTC traits (e.g., glyphosate resistance,glufosinate resistance, imidazolinone resistance, bromoxynil resistance,et al.), and insect resistance traits (Cry1F, Cry1Ab, Cry 34/45, et al.)for example. Additionally, AAD-1 can serve as a selectable marker to aidin selection of primary transformants of plants genetically engineeredwith a second gene or group of genes.

In addition, the subject microbial gene has been redesigned such thatthe protein is encoded by codons having a bias toward both monocot anddicot plant usage (hemicot). Arabidopsis, corn, tobacco, cotton,soybean, canola, and rice have been transformed with AAD-1-containingconstructs and have demonstrated high levels of resistance to both thephenoxy auxin and AOPP herbicides. Thus, the subject invention alsorelates to “plant optimized” genes that encode proteins of the subjectinvention. As shown below in Example 6, the exemplified rebuilt gene wasmore efficacious in conveying herbicide resistance to the plant, ascompared to the bacterial gene.

Oxyalkanoate groups are useful for introducing a stable acidfunctionality into herbicides. The acidic group can impart phloemmobility by “acid trapping,” a desirable attribute for herbicide actionand therefore could be incorporated into new herbicides for mobilitypurposes. Aspects of the subject invention also provide a mechanism ofcreating HTCs. There exist many potential commercial and experimentalherbicides that can serve as substrates for AAD-1. Thus, the use of thesubject genes can also result in herbicide tolerance to those otherherbicides as well.

HTC traits of the subject invention can be used in novel combinationswith other HTC traits (including but not limited to glyphosatetolerance). These combinations of traits give rise to novel methods ofcontrolling weed (and like) species, due to the newly acquiredresistance or inherent tolerance to herbicides (e.g., glyphosate). Thus,in addition to the HTC traits, novel methods for controlling weeds usingherbicides, for which herbicide tolerance was created by said enzyme intransgenic crops, are within the scope of the invention.

Additionally, glyphosate tolerant crops grown worldwide are prevalent.Many times in rotation with other glyphosate tolerant crops, control ofglyphosate-resistant volunteers may be difficult in rotational crops.Thus, the use of the subject transgenic traits, stacked or transformedindividually into crops, provides a tool for controlling other HTCvolunteer crops.

This invention can be applied in the context of commercializing a 2,4-Dresistance trait stacked with current glyphosate resistance traits insoybeans, for example. Thus, this invention provides a tool to combatbroadleaf weed species shifts and/or selection of herbicide resistantbroadleaf weeds, which culminates from extremely high reliance bygrowers on glyphosate for weed control with various crops.

The transgenic expression of the subject AAD-1 genes is exemplified in,for example, Arabidopsis, corn (maize), tobacco, cotton, rice, soybean,and canola. However, the subject invention can be applied to any otherdesired types of plants. Soybeans are a preferred crop fortransformation according to the subject invention. However, thisinvention can be applied to multiple other grass and other broadleafcrops. Likewise, 2,4-D can be more positively utilized in grass cropswhere tolerance to 2,4-D is moderate, and increased tolerance via thistrait would provide growers the opportunity to use 2,4-D at moreefficacious rates and over a wider application timing without the riskof crop injury.

Still further, the subject invention provides a single gene that canprovide resistance to herbicides that control broadleaf weed (auxins)and grass weeds (AOPPs). This gene may be utilized in multiple crops toenable the use of a broad spectrum herbicide combination. The subjectinvention can also control weeds resistant to current chemicals, andaids in the control of shifting weed spectra resulting from currentagronomic practices. The subject AAD-1 can also be used in efforts toeffectively detoxify additional herbicide substrates to non-herbicidalforms. Thus, the subject invention provides for the development ofadditional HTC traits and/or selectable marker technology.

Separate from, or in addition to, using the subject genes to produceHTCs, the subject genes can also be used as selectable markers forsuccessfully selecting transformants in cell cultures, greenhouses, andin the field. There is high inherent value for the subject genes simplyas a selectable marker for biotechnology projects. The promiscuity ofAAD-1 for other phenoxyalkanoic auxinic herbicides provides manyopportunities to utilize this gene for HTC and/or selectable markerpurposes.

One gene of the subject invention, referred to herein as AAD-1(aryloxyalkanoate dioxygenase), was cloned from Sphingobiumherbicidovorans (ATCC 700291) by PCR into pET 280-S/S (designated pDAB3203) and expressed in BL-21 Star E. coli. When this gene isoverexpressed (by induction of 1 mM IPTG and culture lysate combinedwith the following reaction mix: 112.5 μg/ml 2,4-D, 1 mM Ascorbic acid,1 mM α-ketoglutarate, 50 μM Fe(NH₄)₂(SO₄)₂, the recombinantly producedenzyme degrades 2,4-D into herbicidally inactive DCP (as determined byHPLC, mass spectrometry, colorimetric assay, and Arabidopsis plateassay). Additionally, AAD-1 has been demonstrated to convert thefollowing herbicides into their corresponding inactive phenol:dichlorprop, mecoprop, haloxyfop, dichlofop, and others (See Tables 3and 4).

TABLE 3 Effect of purified AAD-1 (v1) on various herbicidal auxins andauxin analogs. Substrates were assayed at 1 mM in 25 mM MOPS pH 6.8, 200μM Fe2+, 200 μM Na ascorbate, 1 mM α-ketoglutarate using either 1 μg or10 μg (10×) purified AAD-1 (v1) per 0.16 ml assay. AAD1 STRUCTURERegistry ID Compound AAD1 (10×)

117613 (R,S)-dichlorprop 0.566 2.594

188874 (R,S)-mecoprop 0.341 2.085

83293 (R,S)-2-chloro, 4- fluorophenoxy- proprionate 0.304 2.358

11113675 (R,S)-3- aminodichlorpop 0.228 2.676

188476 0.077 0.687

192132 0.064 0.204

195517 2,4-D 0.034 0.383

398166 sesone 0.02 0.177

190252 0.008 0.211

124988 0.007 0.058

11263526 0.004 0.069

 

178577 0.003 0.021

178587 0.003 0.02

188527 0.003 0.036

TABLE 4 Effect of purified AAD-1 (v1) on various AOPP graminicides andanalogs, and on cloquintocet. Substrates were assayed at 1 mM in 25 mMMOPS pH 6.8, 200 μM Fe²⁺, 200 μM Na ascorbate, 1 mM α-ketoglutarateusing either 1 μg or 10 μg (10×) purified AAD-1 (v1) per 0.16 ml assayAAD1 STRUCTURE Registry ID Compound AAD1 (10×)

18706 (R)-quizalofop 0.43 2.1

67131 (R,S)-fluazifop 0.427 2.17

11044492 (R)-fenoxaprop 0.408 0.597

34697 (R,S)-clodinofop 0.295 1.98

14603 (R)-cyhalofop 0.222 1.989

14623 (R,S)-cyhalofop 0.215 1.815

62942 (R,S)-fenthiaprop 0.199 1.055

66905 haloxyfop 0.172 1.63

460511 (R,S)-diclofop 0.155 1.663

25646 0.144 1.69

70222 (R,S)-chlorazifop 0.128 1.584

199608 Cyhalofop 0.114 1.26

43865 haloxyfop-oxyacetate 0.004 0.053

7466 (S)-cyhalofop 0.003 0.017

204558 Cloquinotocet 0 0.001

Proteins (and source isolates) of the subject invention. The presentinvention provides functional proteins. By “functional activity” (or“active”) it is meant herein that the proteins/enzymes for use accordingto the subject invention have the ability to degrade or diminish theactivity of a herbicide (alone or in combination with other proteins).Plants producing proteins of the subject invention will preferablyproduce “an effective amount” of the protein so that when the plant istreated with a herbicide, the level of protein expression is sufficientto render the plant completely or partially resistant or tolerant to theherbicide (at a typical rate, unless otherwise specified; typicalapplication rates can be found in the well-known Herbicide Handbook(Weed Science Society of America, Eighth Edition, 2002), for example).The herbicide can be applied at rates that would normally kill thetarget plant, at normal field use rates and concentrations. (Because ofthe subject invention, the level and/or concentration can optionally behigher than those that were previously used.) Preferably, plant cellsand plants of the subject invention are protected against growthinhibition or injury caused by herbicide treatment. Transformed plantsand plant cells of the subject invention are preferably renderedresistant or tolerant to an herbicide, as discussed herein, meaning thatthe transformed plant and plant cells can grow in the presence ofeffective amounts of one or more herbicides as discussed herein.Preferred proteins of the subject invention have catalytic activity tometabolize one or more aryloxyalkanoate compounds.

Transfer of the functional activity to plant or bacterial systems caninvolve a nucleic acid sequence, encoding the amino acid sequence for aprotein of the subject invention, integrated into a protein expressionvector appropriate to the host in which the vector will reside. One wayto obtain a nucleic acid sequence encoding a protein with functionalactivity is to isolate the native genetic material from the bacterialspecies which produce the protein of interest, using information deducedfrom the protein's amino acid sequence, as disclosed herein. The nativesequences can be optimized for expression in plants, for example, asdiscussed in more detail below. Optimized polynucleotide can also bedesigned based on the protein sequence.

The subject invention provides classes of proteins having novelactivities as identified herein. One way to characterize these classesof proteins and the polynucleotides that encode them is by defining apolynucleotide by its ability to hybridize, under a range of specifiedconditions, with an exemplified nucleotide sequence (the complementthereof and/or a probe or probes derived from either strand) and/or bytheir ability to be amplified by PCR using primers derived from theexemplified sequences.

There are a number of methods for obtaining proteins for use accordingto the subject invention. For example, antibodies to the proteinsdisclosed herein can be used to identify and isolate other proteins froma mixture of proteins. Specifically, antibodies may be raised to theportions of the proteins that are most conserved or most distinct, ascompared to other related proteins. These antibodies can then be used tospecifically identify equivalent proteins with the characteristicactivity by immunoprecipitation, enzyme linked immunosorbent assay(ELISA), or immuno-blotting. Antibodies to the proteins disclosedherein, or to equivalent proteins, or to fragments of these proteins,can be readily prepared using standard procedures. Such antibodies arean aspect of the subject invention. Antibodies of the subject inventioninclude monoclonal and polyclonal antibodies, preferably produced inresponse to an exemplified or suggested protein.

One skilled in the art would readily recognize that proteins (and genes)of the subject invention can be obtained from a variety of sources.Since entire herbicide degradation operons are known to be encoded ontransposable elements such as plasmids, as well as genomicallyintegrated, proteins of the subject invention can be obtained from awide variety of microorganisms, for example, including recombinantand/or wild-type bacteria. Other members of the orders Firmicutes andProteobacteria, and specific genera with known rdpA's, such asSphingobium, Delftia, Rodoferax, and Comamonas for example, can be usedas source isolates.

Mutants of bacterial isolates can be made by procedures that are wellknown in the art. For example, asporogenous mutants can be obtainedthrough ethylmethane sulfonate (EMS) mutagenesis of an isolate. Themutants can be made using ultraviolet light and nitrosoguanidine byprocedures well known in the art.

A protein “from” or “obtainable from” any of the subject isolatesreferred to or suggested herein means that the protein (or a similarprotein) can be obtained from the isolate or some other source, such asanother bacterial strain or a plant. “Derived from” also has thisconnotation, and includes proteins obtainable from a given type ofbacterium that are modified for expression in a plant, for example. Oneskilled in the art will readily recognize that, given the disclosure ofa bacterial gene and protein, a plant can be engineered to produce theprotein. Antibody preparations, nucleic acid probes (DNA, RNA, or PNA,for example), and the like can be prepared using the polynucleotideand/or amino acid sequences disclosed herein and used to screen andrecover other related genes from other (natural) sources.

Standard molecular biology techniques may be used to clone and sequencethe proteins and genes described herein. Additional information may befound in Sambrook et al., 1989, which is incorporated herein byreference.

Polynucleotides and probes. The subject invention further providesnucleotide sequences that encode proteins for use according to thesubject invention. The subject invention further provides methods ofidentifying and characterizing genes that encode proteins having thedesired herbicidal activity. In one embodiment, the subject inventionprovides unique nucleotide sequences that are useful as hybridizationprobes and/or primers for PCR techniques. The primers producecharacteristic gene fragments that can be used in the identification,characterization, and/or isolation of specific genes of interest. Thenucleotide sequences of the subject invention encode proteins that aredistinct from previously described proteins.

The polynucleotides of the subject invention can be used to formcomplete “genes” to encode proteins or peptides in a desired host cell.For example, as the skilled artisan would readily recognize, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art. Thelevel of gene expression and temporal/tissue specific expression cangreatly impact the utility of the invention. Generally, greater levelsof protein expression of a degradative gene will result in faster andmore complete degradation of a substrate (in this case a targetherbicide). Promoters will be desired to express the target gene at highlevels unless the high expression has a consequential negative impact onthe health of the plant. Typically, one would wish to have the AAD-1gene constitutively expressed in all tissues for complete protection ofthe plant at all growth stages. However, one could alternatively use avegetatively expressed resistance gene; this would allow use of thetarget herbicide in-crop for weed control and would subsequently controlsexual reproduction of the target crop by application during theflowering stage.

As the skilled artisan knows, DNA typically exists in a double-strandedform. In this arrangement, one strand is complementary to the otherstrand and vice versa. As DNA is replicated in a plant (for example),additional complementary strands of DNA are produced. The “codingstrand” is often used in the art to refer to the strand that binds withthe anti-sense strand. The mRNA is transcribed from the “anti-sense”strand of DNA. The “sense” or “coding” strand has a series of codons (acodon is three nucleotides that can be read as a three-residue unit tospecify a particular amino acid) that can be read as an open readingframe (ORF) to form a protein or peptide of interest. In order toproduce a protein in vivo, a strand of DNA is typically transcribed intoa complementary strand of mRNA which is used as the template for theprotein. Thus, the subject invention includes the use of the exemplifiedpolynucleotides shown in the attached sequence listing and/orequivalents including the complementary strands. RNA and PNA (peptidenucleic acids) that are functionally equivalent to the exemplified DNAmolecules are included in the subject invention.

In one embodiment of the subject invention, bacterial isolates can becultivated under conditions resulting in high multiplication of themicrobe. After treating the microbe to provide single-stranded genomicnucleic acid, the DNA can be contacted with the primers of the inventionand subjected to PCR amplification. Characteristic fragments of genes ofinterest will be amplified by the procedure, thus identifying thepresence of the gene(s) of interest.

Further aspects of the subject invention include genes and isolatesidentified using the methods and nucleotide sequences disclosed herein.The genes thus identified can encode herbicidal resistance proteins ofthe subject invention.

Proteins and genes for use according to the subject invention can beidentified and obtained by using oligonucleotide probes, for example.These probes are detectable nucleotide sequences that can be detectableby virtue of an appropriate label or may be made inherently fluorescentas described in International Application No. WO 93/16094. The probes(and the polynucleotides of the subject invention) may be DNA, RNA, orPNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T),and uracil (U; for RNA molecules), synthetic probes (andpolynucleotides) of the subject invention can also have inosine (aneutral base capable of pairing with all four bases; sometimes used inplace of a mixture of all four bases in synthetic probes) and/or othersynthetic (non-natural) bases. Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “N” or “n” is usedgenerically, “N” or “n” can be G, A, T, C, or inosine. Ambiguity codesas used herein are in accordance with standard IUPAC naming conventionsas of the filing of the subject application (for example, R means A orG, Y means C or T, etc.).

As is well known in the art, if a probe molecule hybridizes with anucleic acid sample, it can be reasonably assumed that the probe andsample have substantial homology/similarity/identity. Preferably,hybridization of the polynucleotide is first conducted followed bywashes under conditions of low, moderate, or high stringency bytechniques well-known in the art, as described in, for example, Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170. For example, as stated therein, low stringency conditionscan be achieved by first washing with 2×SSC (Standard SalineCitrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at roomtemperature. Two washes are typically performed. Higher stringency canthen be achieved by lowering the salt concentration and/or by raisingthe temperature. For example, the wash described above can be followedby two washings with 0.1×SSC/0.1% SDS for 15 minutes each at roomtemperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30minutes each at 55° C. These temperatures can be used with otherhybridization and wash protocols set forth herein and as would be knownto one skilled in the art (SSPE can be used as the salt instead of SSC,for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared bycombining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), andwater, adjusting pH to 7.0 with 10 N NaOH, then adjusting the volume to1 liter. 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml ofautoclaved water, then diluting to 100 ml.

Detection of the probe provides a means for determining in a knownmanner whether hybridization has been maintained. Such a probe analysisprovides a rapid method for identifying genes of the subject invention.The nucleotide segments used as probes according to the invention can besynthesized using a DNA synthesizer and standard procedures. Thesenucleotide sequences can also be used as PCR primers to amplify genes ofthe subject invention.

Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein. That is, one way to define a gene (and the protein it encodes),for example, is by its ability to hybridize (under any of the conditionsspecifically disclosed herein) with a known or specifically exemplifiedgene.

As used herein, “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes can be performed by standardmethods (see, e.g., Maniatis et al. 1982). In general, hybridization andsubsequent washes can be carried out under conditions that allow fordetection of target sequences. For double-stranded DNA gene probes,hybridization can be carried out overnight at 20-25° C. below themelting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz et al. 1983):

Tm=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes can typically be carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at Tm−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes can be determined by the following formula:

Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)

(Suggs et al., 1981).

Washes can typically be out as follows:

-   -   (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

-   -   Low: 1 or 2×SSPE, room temperature    -   Low: 1 or 2×SSPE, 42° C.    -   Moderate: 0.2× or 1×SSPE, 65° C.    -   High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

PCR technology. Polymerase Chain Reaction (PCR) is a repetitive,enzymatic, primed synthesis of a nucleic acid sequence. This procedureis well known and commonly used by those skilled in this art (seeMullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki etal., 1985). PCR is based on the enzymatic amplification of a DNAfragment of interest that is flanked by two oligonucleotide primers thathybridize to opposite strands of the target sequence. The primers arepreferably oriented with the 3′ ends pointing towards each other.Repeated cycles of heat denaturation of the template, annealing of theprimers to their complementary sequences, and extension of the annealedprimers with a DNA polymerase result in the amplification of the segmentdefined by the 5′ ends of the PCR primers. The extension product of eachprimer can serve as a template for the other primer, so each cycleessentially doubles the amount of DNA fragment produced in the previouscycle. This results in the exponential accumulation of the specifictarget fragment, up to several million-fold in a few hours. By using athermostable DNA polymerase such as Taq polymerase, isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other enzymes which can be used are known tothose skilled in the art.

Exemplified DNA sequences, or segments thereof, can be used as primersfor PCR amplification. In performing PCR amplification, a certain degreeof mismatch can be tolerated between primer and template. Therefore,mutations, deletions, and insertions (especially additions ofnucleotides to the 5′ end) of the exemplified primers fall within thescope of the subject invention. Mutations, insertions, and deletions canbe produced in a given primer by methods known to an ordinarily skilledartisan.

Modification of genes and proteins. The subject genes and proteins canbe fused to other genes and proteins to produce chimeric or fusionproteins. The genes and proteins useful according to the subjectinvention include not only the specifically exemplified full-lengthsequences, but also portions, segments and/or fragments (includingcontiguous fragments and internal and/or terminal deletions compared tothe full-length molecules) of these sequences, variants, mutants,chimerics, and fusions thereof. Proteins of the subject invention canhave substituted amino acids so long as they retain desired functionalactivity. “Variant” genes have nucleotide sequences that encode the sameproteins or equivalent proteins having activity equivalent or similar toan exemplified protein. The terms “variant proteins” and “equivalentproteins” refer to proteins having the same or essentially the samebiological/functional activity against the target pests and equivalentsequences as the exemplified proteins. As used herein, reference to an“equivalent” sequence refers to sequences having amino acidsubstitutions, deletions, additions, or insertions that improve or donot adversely affect activity to a significant extent. Fragmentsretaining activity are also included in this definition. Fragments andother equivalents that retain the same or similar function or activityas a corresponding fragment of an exemplified protein are within thescope of the subject invention. Changes, such as amino acidsubstitutions or additions, can be made for a variety of purposes, suchas increasing (or decreasing) protease stability of the protein (withoutmaterially/substantially decreasing the functional activity of theprotein), removing or adding a restriction site, and the like.Variations of genes may be readily constructed using standard techniquesfor making point mutations, for example.

In addition, U.S. Pat. No. 5,605,793, for example, describes methods forgenerating additional molecular diversity by using DNA reassembly afterrandom or focused fragmentation. This can be referred to as gene“shuffling,” which typically involves mixing fragments (of a desiredsize) of two or more different DNA molecules, followed by repeatedrounds of renaturation. This can improve the activity of a proteinencoded by a starting gene. The result is a chimeric protein havingimproved activity, altered substrate specificity, increased enzymestability, altered stereospecificity, or other characteristics.

“Shuffling” can be designed and targeted after obtaining and examiningthe atomic 3D (three dimensional) coordinates and crystal structure of aprotein of interest. Thus, “focused shuffling” can be directed tocertain segments of a protein that are ideal for modification, such assurface-exposed segments, and preferably not internal segments that areinvolved with protein folding and essential 3D structural integrity.

Variant genes can be used to produce variant proteins; recombinant hostscan be used to produce the variant proteins. Using these “geneshuffling” techniques, equivalent genes and proteins can be constructedthat comprise any 5, 10, or 20 contiguous residues (amino acid ornucleotide) of any sequence exemplified herein. As one skilled in theart knows, the gene shuffling techniques, for example, can be adjustedto obtain equivalents having, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, or297 contiguous residues (amino acid or nucleotide), corresponding to asegment (of the same size) in any of the exemplified or suggestedsequences (or the complements (full complements) thereof). Similarlysized segments, especially those for conserved regions, can also be usedas probes and/or primers.

Fragments of full-length genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes that encode active fragments may be obtained using a varietyof restriction enzymes. Proteases may be used to directly obtain activefragments of these proteins.

It is within the scope of the invention as disclosed herein thatproteins can be truncated and still retain functional activity. By“truncated protein” it is meant that a portion of a protein may becleaved off while the remaining truncated protein retains and exhibitsthe desired activity after cleavage. Cleavage can be achieved by variousproteases. Furthermore, effectively cleaved proteins can be producedusing molecular biology techniques wherein the DNA bases encoding saidprotein are removed either through digestion with restrictionendonucleases or other techniques available to the skilled artisan.After truncation, said proteins can be expressed in heterologous systemssuch as E. coli, baculoviruses, plant-based viral systems, yeast, andthe like and then placed in insect assays as disclosed herein todetermine activity. It is well-known in the art that truncated proteinscan be successfully produced so that they retain functional activitywhile having less than the entire, full-length sequence. For example,B.t. proteins can be used in a truncated (core protein) form (see, e.g.,Höfte et al. (1989), and Adang et al. (1985)). As used herein, the term“protein” can include functionally active truncations.

In some cases, especially for expression in plants, it can beadvantageous to use truncated genes that express truncated proteins.Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the full-length protein.

Certain proteins of the subject invention have been specificallyexemplified herein. As these proteins are merely exemplary of theproteins of the subject invention, it should be readily apparent thatthe subject invention comprises variant or equivalent proteins (andnucleotide sequences coding for equivalents thereof) having the same orsimilar activity of the exemplified proteins. Equivalent proteins willhave amino acid similarity (and/or homology) with an exemplifiedprotein. The amino acid identity will typically be at least 60%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 90%, and can be at least 95%. Preferred proteins ofthe subject invention can also be defined in terms of more particularidentity and/or similarity ranges. For example, the identity and/orsimilarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% as compared to a sequence exemplified or suggested herein.Any number listed above can be used to define the upper and lowerlimits.

Unless otherwise specified, as used herein, percent sequence identityand/or similarity of two nucleic acids is determined using the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul 1993.Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12. Gapped BLAST can be used asdescribed in Altschul et al., 1997. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) are used. See NCBI/NIH website. To obtain gappedalignments for comparison purposes, the AlignX function of Vector NTISuite 8 (InforMax, Inc., North Bethesda, Md., U.S.A.), was usedemploying the default parameters. These were: a Gap opening penalty of15, a Gap extension penalty of 6.66, and a Gap separation penalty rangeof 8.

Various properties and three-dimensional features of the protein canalso be changed without adversely affecting the activity/functionalityof the protein. Conservative amino acid substitutions can betolerated/made to not adversely affect the activity and/orthree-dimensional configuration of the molecule. Amino acids can beplaced in the following classes: non-polar, uncharged polar, basic, andacidic. Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution is not adverse tothe biological activity of the compound. Table 5 provides a listing ofexamples of amino acids belonging to each class.

TABLE 5 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made.However, preferred substitutions do not significantly detract from thefunctional/biological activity of the protein.

As used herein, reference to “isolated” polynucleotides and/or“purified” proteins refers to these molecules when they are notassociated with the other molecules with which they would be found innature. Thus, reference to “isolated” and/or “purified” signifies theinvolvement of the “hand of man” as described herein. For example, abacterial “gene” of the subject invention put into a plant forexpression is an “isolated polynucleotide.” Likewise, a protein derivedfrom a bacterial protein and produced by a plant is an “isolatedprotein.”

Because of the degeneracy/redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate alternative DNA sequences that encode the same, or essentiallythe same, proteins. These variant DNA sequences are within the scope ofthe subject invention. This is also discussed in more detail below inthe section entitled “Optimization of sequence for expression inplants.”

Optimization of sequence for expression in plants. To obtain highexpression of heterologous genes in plants it is generally preferred toreengineer the genes so that they are more efficiently expressed in (thecytoplasm of) plant cells. Maize is one such plant where it may bepreferred to re-design the heterologous gene(s) prior to transformationto increase the expression level thereof in said plant. Therefore, anadditional step in the design of genes encoding a bacterial protein isreengineering of a heterologous gene for optimal expression, using codonbias more closely aligned with the target plant sequence, whether adicot or monocot species. Sequences can also be optimized for expressionin any of the more particular types of plants discussed elsewhereherein.

Transgenic hosts. The protein-encoding genes of the subject inventioncan be introduced into a wide variety of microbial or plant hosts. Thesubject invention includes transgenic plant cells and transgenic plants.Preferred plants (and plant cells) are corn, Arabidopsis, tobacco,soybeans, cotton, canola, rice, wheat, turf and pasture grasses, and thelike. Other types of transgenic plants can also be made according to thesubject invention, such as fruits, vegetables, and trees. Moregenerally, dicots and/or monocots can be used in various aspects of thesubject invention.

In preferred embodiments, expression of the gene results, directly orindirectly, in the intracellular production (and maintenance) of theprotein(s) of interest. Plants can be rendered herbicide-resistant inthis manner Such hosts can be referred to as transgenic, recombinant,transformed, and/or transfected hosts and/or cells. In some aspects ofthis invention (when cloning and preparing the gene of interest, forexample), microbial (preferably bacterial) cells can be produced andused according to standard techniques, with the benefit of the subjectdisclosure.

Plant cells transfected with a polynucleotide of the subject inventioncan be regenerated into whole plants. The subject invention includescell cultures including tissue cell cultures, liquid cultures, andplated cultures. Seeds produced by and/or used to generate plants of thesubject invention are also included within the scope of the subjectinvention. Other plant tissues and parts are also included in thesubject invention. The subject invention likewise includes methods ofproducing plants or cells comprising a polynucleotide of the subjectinvention. One preferred method of producing such plants is by plantinga seed of the subject invention.

Insertion of genes to form transgenic hosts. One aspect of the subjectinvention is the transformation/transfection of plants, plant cells, andother host cells with polynucleotides of the subject invention thatexpress proteins of the subject invention. Plants transformed in thismanner can be rendered resistant to a variety of herbicides withdifferent modes of action.

A wide variety of methods are available for introducing a gene encodinga desired protein into the target host under conditions that allow forstable maintenance and expression of the gene. These methods are wellknown to those skilled in the art and are described, for example, inU.S. Pat. No. 5,135,867.

Vectors comprising an AAD-1 polynucleotide are included in the scope ofthe subject invention. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly,the sequence encoding the protein can be inserted into the vector at asuitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered by purification away from genomic DNA. Sequence analysis,restriction analysis, electrophoresis, and other biochemical-molecularbiological methods are generally carried out as methods of analysis.After each manipulation, the DNA sequence used can be restrictiondigested and joined to the next DNA sequence. Each plasmid sequence canbe cloned in the same or other plasmids. Depending on the method ofinserting desired genes into the plant, other DNA sequences may benecessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted. The useof T-DNA for the transformation of plant cells has been intensivelyresearched and described in EP 120 516; Hoekema (1985); Fraley et al.(1986); and An et al. (1985).

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), silicon carbide whiskers, aerosol beaming, PEG, orelectroporation as well as other possible methods. If Agrobacteria areused for the transformation, the DNA to be inserted has to be clonedinto special plasmids, namely either into an intermediate vector or intoa binary vector. The intermediate vectors can be integrated into the Tior Ri plasmid by homologous recombination owing to sequences that arehomologous to sequences in the T-DNA. The Ti or Ri plasmid alsocomprises the vir region necessary for the transfer of the T-DNA.Intermediate vectors cannot replicate themselves in Agrobacteria. Theintermediate vector can be transferred into Agrobacterium tumefaciens bymeans of a helper plasmid (conjugation). Binary vectors can replicatethemselves both in E. coli and in Agrobacteria. They comprise aselection marker gene and a linker or polylinker which are framed by theright and left T-DNA border regions. They can be transformed directlyinto Agrobacteria (Holsters, 1978). The Agrobacterium used as host cellis to comprise a plasmid carrying a vir region. The vir region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be contained. The bacterium so transformed is used for thetransformation of plant cells. Plant explants can be cultivatedadvantageously with Agrobacterium tumefaciens or Agrobacteriumrhizogenes for the transfer of the DNA into the plant cell. Whole plantscan then be regenerated from the infected plant material (for example,pieces of leaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted DNA. No special demands aremade of the plasmids in the case of injection and electroporation. It ispossible to use ordinary plasmids, such as, for example, pUCderivatives.

The transformed cells grow inside the plants in the usual manner Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In some preferred embodiments of the invention, genes encoding thebacterial protein are expressed from transcriptional units inserted intothe plant genome. Preferably, said transcriptional units are recombinantvectors capable of stable integration into the plant genome and enableselection of transformed plant lines expressing mRNA encoding theproteins.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there (and does not come out again). It normallycontains a selection marker that confers on the transformed plant cellsresistance to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, or chloramphenicol, inter alia. Plant selectablemarkers also typically can provide resistance to various herbicides suchas glufosinate, (PAT), glyphosate (EPSPS), imazethyapyr (AHAS), and manyothers. The individually employed marker should accordingly permit theselection of transformed cells rather than cells that do not contain theinserted DNA. The gene(s) of interest are preferably expressed either byconstitutive or inducible promoters in the plant cell. Once expressed,the mRNA is translated into proteins, thereby incorporating amino acidsof interest into protein. The genes encoding a protein expressed in theplant cells can be under the control of a constitutive promoter, atissue-specific promoter, or an inducible promoter.

Several techniques exist for introducing foreign recombinant vectorsinto plant cells, and for obtaining plants that stably maintain andexpress the introduced gene. Such techniques include the introduction ofgenetic material coated onto microparticles directly into cells (U.S.Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco,now Dow AgroSciences, LLC). In addition, plants may be transformed usingAgrobacterium technology, see U.S. Pat. No. 5,177,010 to University ofToledo; U.S. Pat. No. 5,104,310 to Texas A&M; European PatentApplication 0131624B1; European Patent Applications 120516, 159418B1 and176,112 to Schilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763and 4,940,838 and 4,693,976 to Schilperoot; European Patent Applications116718, 290799, 320500, all to Max Planck; European Patent Applications604662 and 627752, and U.S. Pat. No. 5,591,616, to Japan Tobacco;European Patent Applications 0267159 and 0292435, and U.S. Pat. No.5,231,019, all to Ciba Geigy, now Syngenta; U.S. Pat. Nos. 5,463,174 and4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and 5,159,135,both to Agracetus. Other transformation technology includes whiskerstechnology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca,now Syngenta. Electroporation technology has also been used to transformplants. See WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos.5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO93/21335, both to Plant Genetic Systems. Furthermore, viral vectors canalso be used to produce transgenic plants expressing the protein ofinterest. For example, monocotyledonous plants can be transformed with aviral vector using the methods described in U.S. Pat. No. 5,569,597 toMycogen Plant Science and Ciba-Geigy (now Syngenta), as well as U.S.Pat. Nos. 5,589,367 and 5,316,931, both to Biosource, now Large ScaleBiology.

As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod that provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria which are knownto those skilled in the art. The manner of vector transformation intothe Agrobacterium host is not critical to this invention. The Ti or Riplasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al. (1980) and EPO 0 120 515. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is notessential to this invention, with the preferred marker depending on thehost and construction used.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime to allow transformation thereof. After transformation, theAgrobacteria are killed by selection with the appropriate antibiotic andplant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encouraged by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a bacterialprotein is preferably incorporated into a gene transfer vector adaptedto express said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

In addition to numerous technologies for transforming plants, the typeof tissue that is contacted with the foreign genes may vary as well.Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue, tissues for expression in phloem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques described herein.

As mentioned above, a variety of selectable markers can be used, ifdesired. Preference for a particular marker is at the discretion of theartisan, but any of the following selectable markers may be used alongwith any other gene not listed herein which could function as aselectable marker. Such selectable markers include but are not limitedto aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II)which encodes resistance to the antibiotics kanamycin, neomycin andG418, as well as those genes which encode for resistance or tolerance toglyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos orglufosinate); imidazolinones, sulfonylureas and triazolopyrimidineherbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes that are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in Weising et al., 1988. Preferredreporter genes include the beta-glucuronidase (GUS) of the uidA locus ofE. coli, the chloramphenicol acetyl transferase gene from Tn9 of E.coli, the green fluorescent protein from the bioluminescent jellyfishAequorea victoria, and the luciferase genes from firefly Photinuspyralis. An assay for detecting reporter gene expression may then beperformed at a suitable time after said gene has been introduced intorecipient cells. A preferred such assay entails the use of the geneencoding beta-glucuronidase (GUS) of the uidA locus of E. coli asdescribed by Jefferson et al., (1987) to identify transformed cells.

In addition to plant promoter regulatory elements, promoter regulatoryelements from a variety of sources can be used efficiently in plantcells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially Example 7E) and the like may be used. Plantpromoter regulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,heat-shock promoters, and tissue specific promoters. Other elements suchas matrix attachment regions, scaffold attachment regions, introns,enhancers, polyadenylation sequences and the like may be present andthus may improve the transcription efficiency or DNA integration. Suchelements may or may not be necessary for DNA function, although they canprovide better expression or functioning of the DNA by affectingtranscription, mRNA stability, and the like. Such elements may beincluded in the DNA as desired to obtain optimal performance of thetransformed DNA in the plant. Typical elements include but are notlimited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coatprotein leader sequence, osmotin UTR sequences, the maize streak viruscoat protein leader sequence, as well as others available to a skilledartisan. Constitutive promoter regulatory elements may also be usedthereby directing continuous gene expression in all cells types and atall times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissuespecific promoter regulatory elements are responsible for geneexpression in specific cell or tissue types, such as the leaves or seeds(e.g., zein, oleosin, napin, ACP, globulin and the like) and these mayalso be used.

Promoter regulatory elements may also be active (or inactive) during acertain stage of the plant's development as well as active in planttissues and organs. Examples of such include but are not limited topollen-specific, embryo-specific, corn-silk-specific,cotton-fiber-specific, root-specific, seed-endosperm-specific, orvegetative phase-specific promoter regulatory elements and the like.Under certain circumstances it may be desirable to use an induciblepromoter regulatory element, which is responsible for expression ofgenes in response to a specific signal, such as: physical stimulus (heatshock genes), light (RUBP carboxylase), hormone (Em), metabolites,chemical (tetracycline responsive), and stress. Other desirabletranscription and translation elements that function in plants may beused. Numerous plant-specific gene transfer vectors are known in theart.

Plant RNA viral based systems can also be used to express bacterialprotein. In so doing, the gene encoding a protein can be inserted intothe coat promoter region of a suitable plant virus which will infect thehost plant of interest. The protein can then be expressed thus providingprotection of the plant from herbicide damage. Plant RNA viral basedsystems are described in U.S. Pat. No. 5,500,360 to Mycogen PlantSciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to Biosource,now Large Scale Biology.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Method for Identifying Genes that Impart Resistance to 2,4-Din Planta

As a way to identify genes which possess herbicide degrading activitiesin planta, it is possible to mine current public databases such as NCBI(National Center for Biotechnology Information). To begin the process,it is necessary to have a functional gene sequence already identifiedthat encodes a protein with the desired characteristics (i.e.,α-ketoglutarate dioxygenase activity). This protein sequence is thenused as the input for the BLAST (Basic Local Alignment Search Tool)(Altschul et al., 1997) algorithm to compare against available NCBIprotein sequences deposited. Using default settings, this search returnsupwards of 100 homologous protein sequences at varying levels. Theserange from highly identical (85-98%) to very low identity (23-32%) atthe amino acid level. Traditionally only sequences with high homologywould be expected to retain similar properties to the input sequence. Inthis case we chose only those sequences with <50% homology. We go on toexemplify that cloning and recombinantly expressing homologues with aslittle as 27% amino acid conservation can be used to impart commerciallevels of resistance not only to the intended herbicide, but also tosubstrates never previously tested with these enzymes.

PCR and cloning of gene into pET. A single gene (rdpA) was identifiedfrom the NCBI database (see the ncbi.nlm.nih.gov website; accession#AF516752) as a homologue with only 28% amino acid identity to tfdA fromRalstonia eutropha. Percent identity was determined by first translatingboth the rdpA and tfdA DNA sequences deposited in the database toproteins, then using ClustalW in the VectorNTI software package toperform the multiple sequence alignment.

The strain of Sphingobium herbicidovorans containing the rdpA gene wasobtained from ATCC (American Type Culture Collection strain #700291).The lyophilized strain was revived according to ATCC protocol and storedat −80° C. in 20% glycerol for internal use as Dow Bacterial strain DB536. From this freezer stock, a plate of Tryptic Soy Agar was thenstruck out with a loopful of cells for isolation, and incubated at 28°C. for 3 days.

A single colony was used to inoculate 100 ml of Tryptic Soy Broth in a500 ml tri-baffled flask, which was incubated overnight at 28° C. on afloor shaker at 150 rpm. From this, total DNA was isolated with the gramnegative protocol of Qiagen's DNeasy kit (Qiagen cat. #69504). Thefollowing primers were designed to amplify the target gene from genomicDNA, Forward: 5′ TCT AGA AGG AGA TAT ACC ATG CAT GCT GCA CTG TCC CCC CTCTCC CAG CG 3′ [(SEQ ID NO:1) (added Xba I restriction site and RibosomeBinding Site (RBS))] and Reverse: 5′ CTC GAG TTA CTA GCG CGC CGG GCG CACGCC ACC GAC CG 3′ [(SEQ ID NO:2)(added extra stop codon and Xho Isite)].

Twenty microliter reactions were set up as follows: MasterMix 8 ea.primer 1 μl (50 pmoles/μl), gDNA 2.5 μl, H₂O 7.5 μl. PCR was thencarried out under the following conditions: 94° C. 45 sec, 52° C. 1.5minute, 72° C. 1.5 minute, for 30 cycles, followed by a final cycle of72° C. 5 minute, using Eppendorf's Master Taq kit (Eppendorf cat. #0032002.250). The resulting ˜1 kb PCR product was cloned into pCR 2.1(Invitrogen cat. #K4550-40) following the included protocol, withchemically competent TOP10F′ E. coli as the host strain, forverification of nucleotide sequence.

Ten of the resulting white colonies were picked into 4 ml Luria Broth+50μg/ml Kanamycin (LB K), and grown overnight at 37° C. with agitation.Plasmids were purified from each culture using Promega Wizard Plus SVkit (Promega cat. #A1460) and following the included protocol.Sequencing was carried out with Beckman CEQ Quick Start Kit (BeckmanCoulter cat. #608120) using M13 Forward (5′ GTA AAA CGA CGG CCA GT 3′)(SEQ ID NO:16) and Reverse (5′ CAG GAA ACA GCT ATG AC 3′) (SEQ ID NO:17)primers, per manufacturers instructions. This gene sequence (SEQ IDNO:3), and its corresponding protein (SEQ ID NO:9) was given a newgeneral designation for internal consistency AAD-1 (v1)(AryloxyAlkanoate Dioxygenase).

Using the restriction enzymes corresponding to the sites added with theprimer linkers (Xba 1, Xho 1) AAD-1 (v1) was cut out of the pCR2.1vector and ligated into a pET 280 streptomycin/spectinomycin resistantvector. Ligated products were then transformed into TOP10F′ E. coli, andplated on to Luria Broth+50 μg/ml Streptomycin & Spectinomycin (LB S/S)agar plates. To differentiate between AAD-1 (v1):pET 280 and pCR2.1:pET280 ligations, approximately 20 isolated colonies were picked into 6 mlof LB S/S, and grown at 37° C. for 4 hours with agitation.

Each culture was then spotted onto LB K plates, which were incubated at37° C. overnight. Colonies that grew on the LB K were assumed to havethe pCR2.1 vector ligated in, and were discarded. Plasmids were isolatedfrom the remaining cultures as before. This expression construct wasgiven the designation pDAB 3203.

Example 2—Expression and Testing

2.1—HPLC Analysis.

Plasmid pDAB 3203 was maintained frozen at −80° C. in TOP10F′ cells(Invitrogen) as Dow Recombinant strain DR 1878. For expression, plasmidDNA purified from TOP10F′ culture using Promega's Wizard kit (Fishercat. #PR-A1460) was transformed into BL-21 Star (DE3) cells (Invitrogencat. #C6010-03) following manufacturer's protocol. After transformation,50 μl of the cells were plated onto LB S/S agar plates and incubatedovernight at 37° C.

The next morning, all colonies from the entire plate were scraped into100 mls LB in a 500 ml tri-baffled flask and incubated at 37° C./200 rpmfor 1 hr. Gene expression was then induced with 1 mM IPTG, and incubatedfor 4 hrs at 30° C./200 rpm. All 100 ml of culture was centrifuged at4000 rpm for 20 mM The supernatants were then discarded, and the pelletswere resuspended in 10 ml of 50 mM MOPS. These were then subjected tothree 45-sec rounds of sonication to lyse the cells. Following this,lysates were centrifuged at 15,000 rpm to remove cell debris. Thesupernatant was pipetted off and stored at 4° C. To check forrecombinant expression, a 20 μl aliquot was run on a 4-20% Tris Glycinegel (Invitrogen cat. #EC60255).

After expression was confirmed, enzyme activity was tested as follows.First, an aliquot of the cell extract was desalted with a PD-10cartridge (Amersham cat. #17-0435-01). This was then used for subsequentherbicide enzyme reactions.

For each reaction, the following were combined: 2,4-D (125 μg/ml),[Ascorbate (1 mM), Ferrous ion (50 μM), α-ketoglutarate (1 mM), in 100mM MOPS], cell extract (100 μl). This reaction was then incubated atroom temp for 30 mM, after which the reaction was stopped with theaddition of 0.1 N HCl until pH was between 2 and 3. Half of the reactionvolume (˜500 μl) was set aside for bioassay, the remaining volume wasorganically extracted using Solid Phase Extraction tubes (Fisher cat.#11-131-6), eluting with 400 μl of Acetonitrile+0.05% TFA.

The extracts were then tested on HPLC for loss of the 2,4-D peak orpresence of any additional peaks resulting from the degradation ormodification of 2,4-D. Conditions for the HPLC were: Luna 10p, C18(2)250×4.6 mm (Phenomenex cat. #00G-4253-E0), run at 50% ACN+0.05% TFA: 50%H₂O+0.05% TFA to 100% ACN+0.05% TFA over 5 min.

2.2—Plate Test Bioassays for Herbicide Degradation.

Plant bioassays were used to determine if in vitro enzymatic herbicidetransformation resulted in a concomitant loss in herbicidal activity.Because of the selective nature of the herbicides being tested (i.e.,monocot plants controlled by AOPP herbicides and dicot plants controlledby auxinic herbicides), wildtype Agrostis palustris var. Pencross andArabidopsis thaliana var. Columbia were used as monocot and dicot testspecies, respectively. Each species is amenable to germination andgrowth in small Petri dishes.

Arabidopsis seeds were surface sterilized for 10 mM in 50% commercialbleach/deionized water (v/v) with 1 μL of Tween-20 added as a wettingagent with vigorous agitation (shaker table @ 250 rpm). Bleach solutionwas decanted inside a sterile hood and rinsed three times with sterilewater. Bentgrass seeds were surface sterilized for 20 minutes in a likemanner.

Twenty to thirty sterilized seeds for each test species used were addedonto a sterile, solidified agar Plate Test Medium (PTM) [2.5 mM KNO₃,2.5 mM KH₂PO₄, 50 mM FeSO₄, 10 mM NaEDTA (pH 8.0), 2 mM MgSO₄, 2 mMCa(NO₃)₂, 70 μM H₃BO₃, 14 μM MnCl₂, 0.5 μM CuSO₄, 1 μM ZnSO₄, 0.2 μMNaMoO₄.2H₂O, 10 μM NaCl, 10 nM CoCl₂.H₂O, 0.8% (w/v) sucrose, 0.4%agarose (w/v)] for bioassay in 60×15-mm Petri dishes (Falcon 1007). PTMwas additionally modified by adding up to six rates of test herbicidestandards or herbicide-enzyme test solution dilutions such that thefour-fold concentration increments covered a rate range of three ordersof magnitude with the GR₅₀ rate (50% growth reduction) approximately inthe center of the range.

For herbicide-enzyme test solutions, the maximal concentration wasdetermined based on the nominal concentration before any subsequentenzymatic degradation would occur. Seeds were evenly spread by adding 3ml of melted PTM of the same composition, swirling, and allowing tosolidify. Plates were sealed and maintained under sterile conditions ina low light growth chamber (24 h day⁻¹, 100 μE/m²s¹, 23° C.) for 7 days.Root length or root+shoot length were measured for five randomly chosenArabidopsis and bentgrass plants, respectively, average mean length(percent of untreated control) vs. nominal herbicide concentration andGR₅₀ determined.

This bioassay was used to confirm the loss of herbicidal activity as aresult of AAD-1 (v1) degradation of the oxyalkanoate side chain fromvarious agronomically relevant herbicides. In several instances, theanticipated phenol product co-eluted with the parent acid on HPLC andthe bioassay served as the primary screen for herbicide degradation.Tables 6 and 7 represent herbicidal substrates tested.

TABLE 6 Arabidopsis plate test bioassay for commercial phenoxy andpyridinyloxyalkanoate auxin substrates. GR₅₀ (nM) Chemical + ChemicalBlank Chemical + GR₅₀ Chemical tested alone Vector * AAD-1 v1 ratio **Structure 2,4-D 22 17 267 16

DCP >1000 nd nd nd

Dichlorprop nd 30 1000 33

Triclopyr 255 1000 1000 1

Fluroxypyr 2200 2250 1825 <1

* Blank vector represents of cell lysate treatment where E. coli pETvector had no gene insert. ** GR50 ratio is a measure of the loss ofherbicidal activity of enzyme-expressing lysate treatment vs blankvector treatments. A number ≥2 is considered the threshold for detectingherbicide activity loss with this assay.

TABLE 7 Bentgrass plate test bioassay for commercialaryloxyphenoxyalkanoate ACCase-inhibiting substrates. GR₅₀ (nM)Chemical + Chemical + Chemical Blank AAD-1 GR₅₀ Chemical tested aloneVector * v1 ratio ** Structure Haloxyfop-RS 28 21 520 25

Diclofop-RS nd 20 130  7

* Blank vector represents of cell lysate treatment where E. coli pETvector had no gene insert. ** GR50 ratio is a measure of the loss ofherbicidal activity of enzyme-expressing lysate treatment vs blankvector treatments. A number ≥2 is considered the threshold for detectingherbicide activity loss with this assay.

2.3—HPLC Results.

From the literature, it was known that dioxygenase enzymes in this classrequire α-ketoglutarate as a co-substrate (for a general scheme, seeFIG. 1) and ferrous ion to bind in the active site. Other experiments inthe literature have shown that the addition of ascorbate increased theenzymatic activity by maintaining the iron in the reduced state, thuspreventing the enzyme for being degraded. Based on this previous work,initial assays were set up under the assumption that the subject enzymewould work in the same way as other members of this general class ofenzyme.

Surprisingly, the initial HPLC results showed the presence of a new peakat 6.1 minute, in addition to a reduced 2,4-D peak at 5.5 min. This newpeak was not present in the control assay. For an initial identificationof the peak at 6.1 minutes, a DCP control was run under our assayconditions and predictably this also eluted at 6.1 minutes. Theformation of this product was confirmed using a colorimetric assay todetect phenols (see example 3.1) as well as mass spectrometry. Asexpected, AAD-1 (v1) carries out a similar reaction as other members ofthis enzyme class. In the bioassay, these same samples were also shownto have an almost complete loss of 2,4-D herbicidal activity in theArabidopsis plate assay (FIG. 2). Regardless of the specific conditionsof the assay (i.e., longer incubations, more enzyme), only 50-75% of the2,4-D could be degraded to DCP as measured by HPLC. In fact, longerinduction of the BL-21 E. coli cells with IPTG only resulted in lessactive enzyme, even though more total recombinant protein was expressed.

After demonstrating degradation of 2,4-D, additional substrates weretested with similar ring substitutions (i.e., oxyacetates andoxypropionates). The first compounds tested were the pyridine analogsfluroxypyr and triclopyr, which are pyridinyloxyacetates. No enzymeactivity was detected on either of these as substrates. Additional testson various analogs of these two pyridinyloxyacetates with either thefluorine or the amino groups removed also were not degraded.Interestingly however, adding a fluorine to the 5 position of 2,4-Dresulted in an almost total loss of enzyme degradation (see next sectionfor additional results).

ACCase inhibitors, haloxyfop and diclofop, were then tested using thesame conditions as with 2,4-D. (The corresponding phenol metabolitesco-eluted with the parent compound under the HPLC conditions used.) Thebioassay results from these samples showed loss of herbicidal activityagainst both haloxyfop (FIG. 3) and diclofop. These results were alsoconfirmed by the colorimetric assay, which was also used to test a widersampling of these compounds.

2.4—Plate Test Bioassays for Herbicide Degradation.

Bioassay tests results corroborated initial HPLC results that indicatedloss of 2,4-D parent following incubation of 2,4-D solutions withunpurified recombinant AAD-1 (v1) extracts (FIG. 2). Additionally, theherbicidal activity of the phenoxypropionic acid, dichlorprop, was alsoeffectively degraded. The ratio of the nominal GR₅₀ for herbicide+enzymesolution versus the herbicide solution alone served as measure of lossof parent herbicide activity resulting from enzyme activity. A ratio of2-3 typically correlated with 50-75% loss of parent herbicide activity(Table 6). Often a GR₅₀ could not be determined following enzymetreatment; de facto, no detectable herbicide activity remained.

The AOPP class of herbicides, too, served as excellent substrates forAAD-1 (v1) as shown by near complete degradation of graminicidalactivity using the bentgrass plate bioassay (FIG. 3 and Table 7). Thesedata are significant in that this is the first reported observation forany members of this class of enzyme to be active on herbicides outsidethe phenoxy auxins. The implications are that this enzyme is promiscuousenough to utilize chemicals with similar phenoxyalkanoate substructureseven though they have completely different modes of action asherbicides.

Example 3—In Vitro Assay of AAD-1 (v1) Activity Via Colorimetric PhenolDetection

3.1—AAD-1 (v1) Assay.

AAD-1 (v1) enzyme activity was measured by colorimetric detection of theproduct phenol using a protocol modified from that of Fukumori andHausinger (1993) (J. Biol. Chem. 268: 24311-24317) to enable deploymentin a 96-well microplate format. The colorimetric assay has beendescribed for use in measuring the activity of dioxygenases cleaving2,4-D and dichlorprop to release the product 2,4-dichlorophenol.However, other phenols could potentially be released from differentaryloxyalkanoate herbicides such as haloxyfop and cyhalofop (see FIG.4). The color yield from several phenols was compared to that of2,4-dichlorophenol using the detection method previously described toascertain which phenol products could be readily detected. Phenols andphenol analogs were tested at a final concentration of 100 μM in 0.15 ml20 mM MOPS pH 6.75 containing 200 μM NH₄(FeSO₄)₂, 200 μM sodiumascorbate. The phenols derived from haloxyfop and cyhalofop hadequivalent color yields to that of 2,4-dichlorophenol and so werereadily detected. Pyridinols derived from fluroxypyr and triclopyrproduced no significant color. The color yield of 2,4-dichlorophenol andthe haloxyfop phenol was linear and proportional to the concentration ofphenol in the assay up to ˜500 μM. A calibration curve performed understandard assay conditions (160 μl final assay volume) indicated that anabsorbance at 510 nm of 1.0 was obtained from 172 μM phenol.

Enzyme assays were performed in a total volume of 0.15 ml 20 mM MOPS pH6.75 containing 200 μM NH₄FeSO₄, 200 μM sodium ascorbate, 1 mMα-ketoglutarate, the appropriate substrate (added from a 100 mM stockmade up in DMSO), and enzyme. Assays were initiated by addition of thearyloxyalkanoate substrate, enzyme or α-ketoglutarate at time zero.After 15 minutes of incubation at 25° C., the reaction was terminated byaddition of 10 μl 100 mM sodium EDTA. Color was developed by addition of15 μl pH 10 buffer (3.09 g boric acid+3.73 g KCl+44 ml 1 N KOH), 1.5 μl2% 4-aminoantipyrine and 1.5 μl 8% potassium ferricyanide. After 10 to20 mM, the absorbance at 510 nm was recorded in a spectrophotometricmicroplate reader. Blanks contained all reagents except for enzyme toaccount for the occasional slight contamination of some of thesubstrates by small amounts of phenols. Later assays were made moreconvenient by consolidating the additions as follows: the reaction wasquenched by addition of 30 μl of a 1:1:1 mix of 50 mM Na EDTA; pH 10buffer and 0.2% 4-aminoantipyrine, then adding 10 μl 0.8% potassiumferricyanide.

3.2—Extraction.

Activity of recombinant AAD-1 (v1) expressed in Escherichia coli. E.coli cell pellets were resuspended in 0.1 M Tris, pH 7.4+1 mg/mllysozyme (5 ml/cells from 250 ml culture; 20 ml/cells from 1 liter) atroom temperature. After about 15 minutes with occasional shaking, thesuspension was frozen in liquid nitrogen then thawed. DNase was added to0.02 mg/ml final concentration and MgCl₂ to 1 mM. After the extract wasno longer viscous, the extract was centrifuged for 15 mM. Thesupernatant was passed over a BioRad 10DG column pre-equilibrated with20 mM MOPS pH 6.75 and the eluant stored in aliquots at −70° C. Assayswere either performed with these unpurified desalted extracts or withpurified enzymes.

A cell pellet from a 250 ml culture of induced E. coli cells expressingpDAB3203 containing the gene encoding AAD-1 (v1) was extracted andassayed using the previously described protocols. The 2,4-D cleavingactivity in the AAD-1 (v1) extract was compared to that from E. colicells expressing a vector without AAD-1 (v1) using 1 mM 2,4-D and isshown in FIG. 5. The amount of 2,4-dichlorophenol formed is clearlyproportional to the amount of extract added to the assay whereas thecontrol extract contains no 2,4-D cleaving activity.

The activity of this extract was tested on four additional herbicides,(R,S)-dichlorprop, (R,S)-mecoprop, (R,S)-haloxyfop and (R,S)-diclofop incomparison to 2,4-D (all at a final concentration of 0.5 mM) using 4 μlof the E. coli extract per assay with a 15 mM assay period. FIG. 6Ashows that AAD-1 (v1) cleaved all five herbicides to yield a phenol withthe relative activity on the substrates beingdichlorprop=mecoprop>diclofop>haloxyfop>2,4-D. Thus AAD-1 (v1) hasactivity on graminicidal aryloxyphenoxypropionate herbicides as well asphenoxy auxins.

The AAD-1 (v1) extract was then tested using racemic (R,S)-haloxyfop,the R enantiomer of haloxyfop and the S-enantiomer of cyhalofop (all at0.5 mM) as potential substrates to ascertain the likely enantiomericspecificity of AAD-1 (v1). The results are shown in FIG. 6B. Theactivity of the enzyme on (R)-haloxyfop was equivalent to that on(R,S)-haloxyfop whereas no activity could be seen on the S-enantiomer ofcyhalofop indicating that AAD-1 (v1) has R specificity on AOPPs.

Example 4—Substrate Specificity of AAD-1 (v1)

4.1—Additional Substrates of AAD-1 (v1).

The substrate specificity of AAD-1 (v1) toward a variety of commercialand experimental herbicides was tested. Purified AAD-1 (v1) was used ateither 1 or 10 μg per 160 μl assay and each substrate was tested at 1 mMwith an assay time of 15 minutes. Table 3 shows the A510 detected afterthe action of AAD-1 (v1) on a variety of aryloxyalkanoate auxinicherbicides and auxin analogs. The best substrate tested was dichlorprop,with mecoprop also being efficiently cleaved. Two otherphenoxypropionates, the 4-fluoro and 3-amino analogs of dichlorprop,were also acted on effectively by AAD-1 (v1). AAD-1 (v1) produced smallamounts of phenol from a variety of phenoxyacetates including 2,4-D. Therelative rates on these substrates are better gauged from the assaysusing the higher amounts (10 μg) of AAD-1 (v1). From these data, 2,4-Dis cleaved by AAD-1 (v1), as are two phenoxyalkylsulfonates, X188476 andX398166 (sesone).

Table 4 shows data for a variety of AOPP graminicide herbicides as AAD1substrates, and also the safener cloquintocet. All the commercial AOPPherbicides tested were effectively cleaved by AAD-1 (v1). This is anunexpected discovery and greatly increases the potential utility of thisenzyme for conferring resistance to a wide variety of graminicidalherbicides in transgenic uses, in addition to auxins. AAD-1 (v1) had thehighest activity on quizalofop (76% of the dichlorprop rate) and lowestactivity on cyhalofop (27% of the quizalofop rate, 21% of thedichlorprop rate). The aryloxyacetate analog of haloxyfop (X043865) wascleaved very slowly with only a small increase in A510 using the higher(10 μg) amount of enzyme. This is consistent with higher activity ofAAD-1 (v1) seen on phenoxypropionates relative to auxin phenoxyacetates.Minimal activity was detected on (S)-cyhalofop indicating that AAD-1(v1) has a significant preference for the R enantiomers ofaryloxypropionate substrates. Similarly, no activity was noted againstthe quinolinoxyacetate safener, cloquintocet, which is consistent withthe observation that AAD-1 (v1) prefers aryloxypropionate substratesover phenoxy auxins.

Substrates X11115427, X124987 and MCPA were tested at 1 mM using 27 μgcrude recombinant AAD-1 (v1) per assay. All three compounds weresubstrates for AAD-1 (v1) but with different relative effectiveness(Table 8). X11115427 was slightly better as a substrate than 2,4-D (125%of the 2,4-D rate) in contrast to the close analog 3-amino-dichlorprop,which is ˜7-fold better than 2,4-D as a substrate (Table 3). The 5-Fsubstitution appears to decrease the effectiveness of X11115427 as asubstrate for AAD-1 (v1). The rates of product formation from5-F-phenoxyacetate and MCPA were 32% and 55% that of 2,4-D respectively.

TABLE 8 Effect of AAD-1 (v1) on three substrates relative to 2,4-D.Substrates were assayed as in Table 6 at 1 mM using a crude recombinantAAD-1 (v1) extract from E. coli. Effect of AAD-1 (v1) on threesubstrates relative to 2,4-D. Registry ID MOLSTRUCTURE Compound A510 %2,4-D 195517

2,4-D 0.177 100 11115427

(R,S)-3-amino, 5- F-dichlorprop 0.221 125 124987

5-F, 2,4-D 0.056  32 192711

MCPA 0.097  55

4.2—Kinetic Characterization.

The K_(m) and k_(cat) values of purified AAD-1 (v1) (see Example 10)were determined for four herbicide substrates, (R)-dichlorprop,(R)-haloxyfop, (R)-quizalofop and 2,4-D under standard assay conditions(25 mM MOPS, pH 6.8; 200 μM Na ascorbate; 200 μM Fe²⁺; 1 mMα-ketoglutarate; 25° C.). The dose response curves were fitted usingGrafit (Erithacus Software, UK), and the graphs and derived constantsare shown in FIG. 7 and Table 9 respectively. The K_(m) values for thefour substrates were fairly similar (75-125 μM), but the k_(cat) valuesvaried significantly. (R)-dichlorprop had the highest k_(cat) value and2,4-D the lowest (10% that of (R)-dichlorprop). These k_(cat) valueswere consistent with the range of values seen in the substratespecificity tests shown in Table 3 and Table 4 as these were performedat high (saturating) substrate concentrations (1 mM).

TABLE 9 Kinetic Constants for AAD-1 (v1) Substrates. Kinetic constantswere derived from the data in FIG. 7 using Grafit fitting to theMichaelis-Menten equation. Table 9. Kinetic Constants for AAD-1 (v1)Substrates. V_(max) K_(m) (μmol min⁻¹ mg⁻¹ k_(cat) k_(cat)/K_(m)Substrate (μM) ± SE AAD1) ± SE (min⁻¹) (min⁻¹ mM⁻¹) R-dichlorprop  75 ±10 0.79 ± 0.03 26.1 348 R-quizalofop 125 ± 20 0.57 ± 0.03 18.9 151R-haloxyfop 120 ± 54 0.34 ± 0.04 11.2 94 2,4-D 96 ± 8 0.57 ± 0.00 2.7 28

The relative K_(m) and k_(cat) values for dichlorprop and 2,4-D differsignificantly from those published for the R-specific dioxygenase fromDelftia acidovorans by Westendorf et al. (2003) (Acta Biotechnol. 23:3-17). The published k_(cat)/K_(m) value for 2,4-D is 0.6% that ofdichlorprop, whereas in our studies, the k_(cat)/K_(m) value for 2,4-Dis 8% that of dichlorprop. Thus, in this study, AAD-1 (v1) isunexpectedly effective at catalyzing the cleavage of 2,4-D. Thisincreases its potential utility for conferring diverse herbicidetolerance traits in transgenic applications.

4.3—Additional Substrates for AAD-1 (v1).

Three additional substrates were tested at 1 mM using 27 μg cruderecombinant AAD-1 (v1) per assay; X11115427, X124987 and MCPA. Theresults are shown in Table 8. All three compounds were substrates forAAD-1 (v1) but with different relative effectiveness. X11115427 was onlyslightly better (125%) as a substrate than 2,4-D. This is in contrast to3-aminodichlorprop which is >7 fold better than 2,4-D as a substrate(Table 3). Thus, the 5-F substitution has significantly decreased theeffectiveness of X11115427 as a substrate for AAD-1 (v1). A similarpattern is seen with 5-F-2,4-D which is only 32% as effective as asubstrate relative to 2,4-D. In this assay, MCPA was also less effectiveas a substrate of AAD-1 (v1) (55% relative to 2,4-D).

Example 5—Optimization of Sequence for Expression in Plants

5.1—Background.

To obtain high expression of heterologous genes in plants, it may bepreferred to reengineer said genes so that they are more efficientlyexpressed in (the cytoplasm of) plant cells. Maize is one such plantwhere it may be preferred to re-design the heterologous gene(s) prior totransformation to increase the expression level thereof in said plant.Therefore, an additional step in the design of genes encoding abacterial protein is reengineering of a heterologous gene for optimalexpression.

One reason for the reengineering of a bacterial protein for expressionin maize is due to the non-optimal G+C content of the native gene. Forexample, the very low G+C content of many native bacterial gene(s) (andconsequent skewing towards high A+T content) results in the generationof sequences mimicking or duplicating plant gene control sequences thatare known to be highly A+T rich. The presence of some A+T-rich sequenceswithin the DNA of gene(s) introduced into plants (e.g., TATA box regionsnormally found in gene promoters) may result in aberrant transcriptionof the gene(s). On the other hand, the presence of other regulatorysequences residing in the transcribed mRNA (e.g., polyadenylation signalsequences (AAUAAA), or sequences complementary to small nuclear RNAsinvolved in pre-mRNA splicing) may lead to RNA instability. Therefore,one goal in the design of genes encoding a bacterial protein for maizeexpression, more preferably referred to as plant optimized gene(s), isto generate a DNA sequence having a higher G+C content, and preferablyone close to that of maize genes coding for metabolic enzymes. Anothergoal in the design of the plant optimized gene(s) encoding a bacterialprotein is to generate a DNA sequence in which the sequencemodifications do not hinder translation.

Table 10 illustrates how high the G+C content is in maize. For the datain Table 10, coding regions of the genes were extracted from GenBank(Release 71) entries, and base compositions were calculated using theMacVector™ program (Accelerys, San Diego, Calif.). Intron sequences wereignored in the calculations.

TABLE 10 Compilation of G + C contents of protein coding regions ofmaize genes Protein Class.sup.a Range % G + C Mean % G + C.sup.bMetabolic Enzymes (76) 44.4-75.3 59.0 (.+−.8.0) Structural Proteins (18)48.6-70.5 63.6 (.+−.6.7) Regulatory Proteins (5) 57.2-68.8 62.0(.+−.4.9) Uncharacterized Proteins (9) 41.5-70.3 64.3 (.+−.7.2) AllProteins (108) 44.4-75.3 60.8 (.+−.5.2) .sup.a Number of genes in classgiven in parentheses. .sup.b Standard deviations given in parentheses..sup.c Combined groups mean ignored in mean calculation

Due to the plasticity afforded by the redundancy/degeneracy of thegenetic code (i.e., some amino acids are specified by more than onecodon), evolution of the genomes in different organisms or classes oforganisms has resulted in differential usage of redundant codons. This“codon bias” is reflected in the mean base composition of protein codingregions. For example, organisms with relatively low G+C contents utilizecodons having A or T in the third position of redundant codons, whereasthose having higher G+C contents utilize codons having G or C in thethird position. It is thought that the presence of “minor” codons withinan mRNA may reduce the absolute translation rate of that mRNA,especially when the relative abundance of the charged tRNA correspondingto the minor codon is low. An extension of this is that the diminutionof translation rate by individual minor codons would be at leastadditive for multiple minor codons. Therefore, mRNAs having highrelative contents of minor codons would have correspondingly lowtranslation rates. This rate would be reflected by subsequent low levelsof the encoded protein.

In engineering genes encoding a bacterial protein for maize (or otherplant, such as cotton or soybean) expression, the codon bias of theplant has been determined. The codon bias for maize is the statisticalcodon distribution that the plant uses for coding its proteins and thepreferred codon usage is shown in Table 11. After determining the bias,the percent frequency of the codons in the gene(s) of interest isdetermined. The primary codons preferred by the plant should bedetermined, as well as the second, third, and fourth choices ofpreferred codons when multiple choices exist. A new DNA sequence canthen be designed which encodes the amino sequence of the bacterialprotein, but the new DNA sequence differs from the native bacterial DNAsequence (encoding the protein) by the substitution of the plant (firstpreferred, second preferred, third preferred, or fourth preferred)codons to specify the amino acid at each position within the proteinamino acid sequence. The new sequence is then analyzed for restrictionenzyme sites that might have been created by the modification. Theidentified sites are further modified by replacing the codons withfirst, second, third, or fourth choice preferred codons. Other sites inthe sequence which could affect transcription or translation of the geneof interest are the exon:intron junctions (5′ or 3′), poly A additionsignals, or RNA polymerase termination signals. The sequence is furtheranalyzed and modified to reduce the frequency of TA or GC doublets. Inaddition to the doublets, G or C sequence blocks that have more thanabout four residues that are the same can affect transcription of thesequence. Therefore, these blocks are also modified by replacing thecodons of first or second choice, etc. with the next preferred codon ofchoice.

TABLE 11 Preferred amino acid codons for proteins expressed in maizeAmino Acid Codon* Alanine GCC/GCG Cysteine TGC/TGT Aspartic Acid GAC/GATGlutamic Acid GAG/GAA Phenylalanine TTC/TTT Glycine GGC/GGG HistidineCAC/CAT Isoleucine ATC/ATT Lysine AAG/AAA Leucine CTG/CTC Methionine ATGAsparagine AAC/AAT Proline CCG/CCA Glutamine CAG/CAA Arginine AGG/CGCSerine AGC/TCC Threonine ACC/ACG Valine GTG/GTC Tryptophan TGG TryrosineTAC/TAT Stop TGA/TAG

It is preferred that the plant optimized gene(s) encoding a bacterialprotein contain about 63% of first choice codons, between about 22% toabout 37% second choice codons, and between about 15% to about 0% thirdor fourth choice codons, wherein the total percentage is 100%. Mostpreferred the plant optimized gene(s) contains about 63% of first choicecodons, at least about 22% second choice codons, about 7.5% third choicecodons, and about 7.5% fourth choice codons, wherein the totalpercentage is 100%. The method described above enables one skilled inthe art to modify gene(s) that are foreign to a particular plant so thatthe genes are optimally expressed in plants. The method is furtherillustrated in PCT application WO 97/13402.

Thus, in order to design plant optimized genes encoding a bacterialprotein, a DNA sequence is designed to encode the amino acid sequence ofsaid protein utilizing a redundant genetic code established from a codonbias table compiled for the gene sequences for the particular plant. Theresulting DNA sequence has a higher degree of codon diversity, adesirable base composition, can contain strategically placed restrictionenzyme recognition sites, and lacks sequences that might interfere withtranscription of the gene, or translation of the product mRNA. Thus,synthetic genes that are functionally equivalent to the proteins/genesof the subject invention can be used to transform hosts, includingplants. Additional guidance regarding the production of synthetic genescan be found in, for example, U.S. Pat. No. 5,380,831.

5.2—Rebuild Analysis.

Extensive analysis of the 888 base pairs (bp) of the DNA sequence of thenative AAD-1 (v1) coding region (SEQ ID NO:3) revealed the presence ofseveral sequence motifs that are thought to be detrimental to optimalplant expression, as well as a non-optimal codon composition. To improveproduction of the recombinant protein in monocots as well as dicots, a“plant-optimized” DNA sequence (SEQ ID NO:5) was developed that encodesSEQ ID NO:11, which is the same as the native SEQ ID NO:9 except for theaddition of an alanine residue at the second position. The additionalalanine codon (GCT; underlined in SEQ ID NO:5) was included to encode anNco I site (CCATGG) spanning the ATG start codon, to enable subsequentcloning operations. The proteins encoded by the native (v1) andplant-optimized (v3) coding regions are 99.3% identical, differing onlyat amino acid number 2. In contrast, the native (v1) and plant-optimized(v3) DNA sequences of the coding regions are only 77.7% identical. Asequence alignment was made of the native and plant-optimized DNAs, andTable 12 shows the differences in codon compositions of the native andplant-optimized sequences.

TABLE 12 Codon composition comparison of native AAD-1 (v1) coding regionand Plant-Optimized version. Amino Native Native Plant Opt Plant OptAmino Native Plant Opt Plant Opt Acid Codon Gene # Gene % Gene # Gene %Acid Codon Gene # Gene # Gene % ALA (A) GCA 2 9.1 5 22 LEU (L) CTA 0 0.00 0 22 GCC 10 45.5 8 35 22 CTC 5 22.7 7 32 GCG 9 40.9 0 0 CTG 16 72.7 00 GCT 1 4.5 10 43 CTT 0 0.0 8 36 ARG (R) AGA 0 0.0 7 30 TTA 0 0.0 0 0 23AGG 0 0.0 7 30 TTG 1 4.5 7 32 CGA 0 0.0 0 0 LYS (K) AAA 1 14.3 2 29 CGC16 69.6 5 22 7 AAG 6 85.7 5 71 CGG 4 17.4 0 0 MET (M) ATG 8 100 8 100CGT 3 13.0 4 17 PHE (F) TTC 10 76.9 9 69 ASN (N) AAC 8 100.0 4 50 13 TTT3 23.1 4 31 8 AAT 0 0.0 4 50 PRO (P) CCA 1 5.9 8 47 ASP (D) GAC 15 78.910 53 17 CCC 9 52.9 1 6 19 GAT 4 21.1 9 47 CCG 7 41.2 0 0 CYS (C) TGC 3100.0 2 67 CCT 0 0.0 8 47 3 TGT 0 0.0 1 33 SER (S) AGC 11 73.3 4 27 ENDTAA 0 0.0 0 0 15 AGT 0 0.0 0 0 1 TAG 1 100.0 0 0 TCA 0 0.0 4 27 TGA 00.0 1 100 TCC 2 13.3 3 20 GLN (Q) CAA 0 0.0 7 54 TCG 2 13.3 0 0 13 CAG13 100.0 6 46 TCT 0 0.0 4 27 GLU (E) GAA 8 50.0 5 31 THR (T) ACA 1 4.3 626 16 GAG 8 50.0 11 69 23 ACC 16 69.6 9 39 GLY (G) GGA 0 0.0 6 29 ACG 521.7 0 0 21 GGC 15 71.4 7 33 ACT 1 4.3 8 35 GGG 3 14.3 2 10 TRP (W) TGG5 100 5 100 GGT 3 14.3 6 29 TYR (Y) TAC 7 70.0 6 60 HIS (H) CAC 6 60.0 550 10 TAT 3 30.0 4 40 10 CAT 4 40.0 5 50 VAL (V) GTA 2 7.1 0 0 ILE (I)ATA 0 0.0 3 25 28 GTC 11 39.3 8 29 12 ATC 12 100.0 5 42 GTG 15 53.6 1036 ATT 0 0.0 4 33 GTT 0 0.0 10 36 Totals 148 149 Totals 148 148

5.3—Completion of Binary Vectors.

5.3.1—Rebuilt AAD-1 (v3). The plant optimized gene AAD-1 (v3) wasreceived from Picoscript (the gene rebuild design was completed (seeabove) and out-sourced to Picoscript for construction) and sequenceverified (SEQ ID NO:5) internally, to confirm that no alterations of theexpected sequence were present. The sequencing reactions were carriedout with M13 Forward (SEQ ID NO:16) and M13 Reverse (SEQ ID NO:17)primers using the Beckman Coulter “Dye Terminator Cycle Sequencing withQuick Start Kit” reagents as before. Sequence data was analyzed andresults indicated that no anomalies were present in the plant optimizedAAD-1 (v3) DNA sequence. The AAD-1 (v3) gene was cloned into pDAB726 asan 0I-Sac I fragment. The resulting construct was designated pDAB720,containing: [AtUbi10 promoter: Nt OSM 5′UTR: AAD-1 (v3): Nt OSM3′UTR:ORF1 polyA3′UTR] (verified with Not I restriction digests). A Not I-NotI fragment containing the described cassette was then cloned into theNot I site of the binary vector pDAB3038. The resulting binary vector,pDAB721, containing the following cassette [AtUbi10 promoter: NtOSM5′UTR: AAD-1 (v3): Nt OSM 3′UTR: ORF1 polyA 3′UTR: CsVMV promoter:PAT: ORF25/26 3′UTR] was restriction digested (with Bam HI, EcoR I, EcoRV, HinD III, Pac I, and Xmn I) for verification of the correctorientation. The verified completed construct (pDAB721) was used fortransformation into Agrobacterium (see section 6.2).

5.3.2—Native AAD1 (v1) and modified AAD-1 (v2). The AAD-1 (v1) gene (SEQID NO:3) was PCR amplified from pDAB3203. During the PCR reaction,alterations were made within the primers to introduce the NcoI and SacIrestriction sites in the 5′ primer and 3′ primer, respectively. Theprimers “rdpA(ncoI)” [CCC ATG GCT GCT GCA CTG TCC CCC CTC TCC] (SEQ IDNO:6) and “3′saci” [GAG CTC ACT AGC GCG CCG GGC GCA CGC CAC CGA] (SEQ IDNO:7) were used to amplify a DNA fragment using the Fail Safe PCR System(Epicenter).

The PCR amplicon was ligated into the pCR 2.1 TOPO TA cloning vector(Invitrogen) and sequence verified with M13 Forward (SEQ ID NO:16) andM13 Reverse (SEQ ID NO:17) primers using the Beckman Coulter “DyeTerminator Cycle Sequencing with Quick Start Kit” sequencing reagents.

Sequence data identified a clone with the correct sequence. Duringanalysis a superfluous NotI restriction site was identified toward the3′ end of AAD-1 (v1). This site was removed to facilitate cloning intopDAB3038. To remove the additional site a PCR reaction was performedwith an internal 5′ primer. The NotI site was altered by incorporating anew codon for an amino acid to remove the spurious NotI site. Thischange would alter the arginine at position 212 to a cysteine. The PCRprimers “BstEII/Del NotI” [TGG TGG TGA CCC ATC CGG GCA GCG GCT GCA AGGGCC] (SEQ ID NO:8) and “3′ saci” (SEQ ID NO:7) were used.

A PCR reaction was completed using the Fail Safe PCR System (Epicenter)and the resulting fragment was cloned into the pCR 2.1 TOPO TA cloningkit (Invitrogen). Confirmation of the correct PCR product was completedby DNA sequencing, and the “fixed” gene was given the designation AAD-1(v2) (SEQ ID NO:4).

A sequencing reaction using the M13 Reverse primer (SEQ ID NO:17) andthe Beckman Coulter “Dye Terminator Cycle Sequencing with Quick StartKit” sequencing reagents indicated that the correct PCR fragment hadbeen isolated. This construct was digested with the BstEII and SacIenzymes. The resulting fragment was cloned into the pCR2.1 AAD-1 (v2)construct (pCR2.1 Delta NotI) and confirmed via restriction enzymedigestion.

The modified AAD-1 (v2) gene was then cloned into pDAB726 as a NcoI/SacIDNA fragment. The resulting construct (pDAB708) was verified withrestriction digests. This construct was then cloned into the binarypDAB3038 as a NotI-NotI fragment. The final resulting construct wasgiven the designation pDAB766, containing the [AtUbi10 promoter: NtOSM5′UTR: AAD-1 (v2): Nt OSM 3′UTR: ORF1 polyA 3′UTR: CsVMV promoter:PAT: ORF25/26 3′UTR] and was restriction digested for verification ofthe correct orientation. The completed construct was then used fortransformation into Agrobacterium.

5.3.3—Design of a soybean-codon-biased DNA sequence encoding a soybeanEPSPS having mutations that confer glyphosate tolerance. This exampleteaches the design of a new DNA sequence that encodes a mutated soybean5-enolpyruvoylshikimate 3-phosphate synthase (EPSPS), but is optimizedfor expression in soybean cells. The amino acid sequence of atriply-mutated soybean EPSPS is disclosed as SEQ ID NO:5 of WO2004/009761. The mutated amino acids in the so-disclosed sequence are atresidue 183 (threonine of native protein replaced with isoleucine),residue 186 (arginine in native protein replaced with lysine), andresidue 187 (proline in native protein replaced with serine). Thus, onecan deduce the amino acid sequence of the native soybean EPSPS proteinby replacing the substituted amino acids of SEQ ID NO:5 of WO2004/009761 with the native amino acids at the appropriate positions.Such native protein sequence is presented herein as SEQ ID NO:20. Adoubly mutated soybean EPSPS protein sequence, containing a mutation atresidue 183 (threonine of native protein replaced with isoleucine), andat residue 187 (proline in native protein replaced with serine) ispresented herein as SEQ ID NO:21.

A codon usage table for soybean (Glycine max) protein coding sequences,calculated from 362,096 codons (approximately 870 coding sequences), wasobtained from the “kazusa.or.jp/codon” World Wide Web site. Those datawere reformatted as displayed in Table 13. Columns D and H of Table 13present the distributions (in % of usage for all codons for that aminoacid) of synonymous codons for each amino acid, as found in the proteincoding regions of soybean genes. It is evident that some synonymouscodons for some amino acids (an amino acid may be specified by 1, 2, 3,4, or 6 codons) are present relatively rarely in soybean protein codingregions (for example, compare usage of GCG and GCT codons to specifyalanine). A biased soybean codon usage table was calculated from thedata in Table 13. Codons found in soybean genes less than about 10% oftotal occurrences for the particular amino acid were ignored. To balancethe distribution of the remaining codon choices for an amino acid, aweighted average representation for each codon was calculated, using theformula:

Weighted % of C1=1/(% C1+% C2+% C3+etc.)×% C1×100

where C1 is the codon in question, C2, C3, etc. represent the remainingsynonymous codons, and the % values for the relevant codons are takenfrom columns D and H of Table 13 (ignoring the rare codon values in boldfont). The Weighted % value for each codon is given in Columns C and Gof Table 13. TGA was arbitrarily chosen as the translation terminator.The biased codon usage frequencies were then entered into a specializedgenetic code table for use by the OptGene™ gene design program (OcimumBiosolutions LLC, Indianapolis, Ind.).

TABLE 13 Synonymous codon representation in soybean protein codingsequences, and calculation of a biased codon representation set forsoybean-optimized synthetic gene design. A C D E G H Amino B WeightedSoybean Amino F Weighted Soybean Acid Codon % % Acid Codon % % ALA (A)GCA 33.1 30.3 LEU (L) CTA DNU 9.1 GCC 24.5 22.5 CTC 22.4 18.1 GCG  DNU*8.5 CTG 16.3 13.2 GCT 42.3 38.7 CTT 31.5 25.5 ARG (R) AGA 36.0 30.9 TTADNU 9.8 AGG 32.2 27.6 TTG 29.9 24.2 CGA DNU 8.2 LYS (K) AAA 42.5 42.5CGC 14.8 12.7 AAG 57.5 57.5 CGG DNU 6.0 MET (M) ATG 100.0  100 CGT 16.914.5 PHE (F) TTC 49.2 49.2 ASN (N) AAC 50.0 50.0 TTT 50.8 50.8 AAT 50.050.0 PRO (P) CCA 39.8 36.5 ASP (D) GAC 38.1 38.1 CCC 20.9 19.2 GAT 61.961.9 CCG DNU 8.3 CYS (C) TGC 50.0 50.0 CCT 39.3 36.0 TGT 50.0 50.0 SER(S) AGC 16.0 15.1 END TAA DNU 40.7 AGT 18.2 17.1 TAG DNU 22.7 TCA 21.920.6 TGA 100.0  36.6 TCC 18.0 16.9 GLN (Q) CAA 55.5 55.5 TCG DNU 6.1 CAG44.5 44.5 TCT 25.8 24.2 GLU (E) GAA 50.5 50.5 THR (T) ACA 32.4 29.7 GAG49.5 49.5 ACC 30.2 27.7 GLY (G) GGA 31.9 31.9 ACG DNU 8.3 GGC 19.3 19.3ACT 37.4 34.3 GGG 18.4 18.4 TRP (W) TGG 100.0  100 GGT 30.4 30.4 TYR (Y)TAC 48.2 48.2 HIS (H) CAC 44.8 44.8 TAT 51.8 51.8 CAT 55.2 55.2 VAL (V)GTA 11.5 11.5 ILE (I) ATA 23.4 23.4 GTC 17.8 17.8 ATC 29.9 29.9 GTG 32.032.0 ATT 46.7 46.7 GTT 38.7 38.7 *DNU = Do Not Use

To derive a soybean-optimized DNA sequence encoding the doubly mutatedEPSPS protein, the protein sequence of SEQ ID NO:21 wasreverse-translated by the OptGene™ program using the soybean-biasedgenetic code derived above. The initial DNA sequence thus derived wasthen modified by compensating codon changes (while retaining overallweighted average representation for the codons) to reduce the numbers ofCG and TA doublets between adjacent codons, increase the numbers of CTand TG doublets between adjacent codons, remove highly stableintrastrand secondary structures, remove or add restriction enzymerecognition sites, and to remove other sequences that might bedetrimental to expression or cloning manipulations of the engineeredgene. Further refinements of the sequence were made to eliminatepotential plant intron splice sites, long runs of A/T or C/G residues,and other motifs that might interfere with RNA stability, transcription,or translation of the coding region in plant cells. Other changes weremade to eliminate long internal Open Reading Frames (frames other than+1). These changes were all made within the constraints of retaining thesoybean-biased codon composition as described above, and whilepreserving the amino acid sequence disclosed as SEQ ID NO:21.

The soybean-biased DNA sequence that encodes the EPSPS protein of SEQ IDNO:21 is given as bases 1-1575 of SEQ ID NO:22. Synthesis of a DNAfragment comprising SEQ ID NO:22 was performed by a commercial supplier(PicoScript, Houston Tex.).

5.3.4—Cloning of additional binary constructs. The completion ofpDAB3295 and pDAB3757 incorporated the use of the GateWay CloningTechnology (Invitrogen, cat #11791-043 and cat #12535-019). The GateWayTechnology uses lambda phage-based site-specific recombination to inserta gene cassette into a vector. For more information refer to GatewayTechnology: A universal technology to clone DNA sequence for functionalanalysis and expression in multiple systems, © 1999-2003, InvitrogenCorp., 1600 Faraday Ave., Carlsbad, Calif. 92008 (printed—2003). Allother constructs created for transformation into appropriate plantspecies were built using similar procedures as above and other standardmolecular cloning methods (Maniatis et al., 1982). Table 14 lists allthe transformation constructs used with appropriate promoters andfeatures defined, as well as the crop transformed.

The sacB gene was added to the binary vector pDAB3289 as a bacterialnegative selection marker to reduce the persistence of Agrobacteriumassociated with transformed plant tissue. SacB is a levan-sucrase enzymeproduced by Bacillus spp. and is toxic to most Gram negative bacteriawhen grown in the presence of sucrose (Gay et al., 1983). The sacB genewas recovered on a Hind III fragment from plasmid pRE112 (Edwards, etal., 1998) and cloned into the unique Hind III site in pDAB3289.

TABLE 14 Binary constructs used in transformation of various plantspecies. Species Gene Bacte- Plant * Trans- of in- Fea- Fea- Bacterialrial Se- Selec- Trxn pDAB pDAS formed terest Pro- ture ture GOI Pro-Selection lection tion Pro- Meth- # # into (GOI) moter 1 2 2 moter genegene 2 gene moter od 721 A, T, AAD1 AtUbi10 NtOsm — — — Erythromycin —pat CsVMV Agro Ct, S, v3 binary Ca 3230 A EPSPS AtUbi10 NtOsm RB7 Mar —— Spectinomycin — AAD1 CsVMV Agro v2 v3 binary 3289 S AAD1 CsVMV NtOsmRB7 Mar EPSPS AtUbi10 Spectinomycin sacB HptII AtUbi3 Agro v3 v2 binary3291 S AAD1 CsVMV NtOsm RB7 Mar EPSPS AtUbi10 Spectinomycin — HptIIAtUbi3 Agro v3 v2 binary 3295 S AAD1 CsVMV NtOsm RB7 Mar — —Spectinomycin — pat AtUbi10 Aerosol v3 v2 beam 3297 1270 A, T AAD1 CsVMVNtOsm RB7 Mar — — Spectinomycin — pat AtUbi10 Agro v3 v2 binary 3403 Cn,R AAD1 ZmUbi1 — RB7 Mar — — Ampicillin — Same as GOI Whis- v3 v2 kers/Gun 3404 Cn AAD1 ZmUbi1 — RB7 Mar — — Ampicillin — pat OsAct1 Whis- v3v2 kers 3415 1283 Cn AAD1 ZmUbi1 — RB7 Mar — — Ampicillin — AHAS OsAct1Whis- v3 v2 v3 kers 3602 1421 Cn AAD1 ZmUbi1 — RB7 Mar — — Spectinomycin— AHAS OsAct1 Agro v3 v2 v3 Super- binary 3757 Ca AAD1 CsVMV NtOsm RB7Mar EPSPS AtUbi10 Spectinomycin — pat AtUbi11 Agro v3 v2 binary 3705 AAAD2 AtUbi10 NtOsm RB7 Mar — — Erythromycin — pat CsVMV Agro v2 v2binary * A = Arabidopsis T = Tobacco S = Soybean Ct = Cotton R = Rice Cn= Corn Ca = Canola CsVMV = Cassava Vein Mosaic Virus Promoter AtUbi10 =Arabidopsis thaliana Ubiquitin 10 Promoter RB7 Mar v2 = Nicotianatabacum matrix associated region (MAR) Nt Osm = Nicotiana tabacumOsmotin 5′ Untranslated Region and the Nicotiana tabacum Osmotin 3′Untranslated Region (721 and 793) Atu ORF1 3′ UTR = Agrobacteriumtumefaciens Open Reading Frame 1 3′ Untranslated Region (3295 and 3757)Atu ORF24 3′ UTR = Agrobacterium tumefaciens Open Reading Frame 24 3′Untranslated Region ZmUbi1 = Zea mays Ubiquitin 1 Promoter HptII =hygromycin phosphotransferase

Example 6—Transformation into Arabidopsis and Selection

6.1—Arabidopsis thaliana Growth Conditions.

Wildtype Arabidopsis seed was suspended in a 0.1% Agarose (SigmaChemical Co., St. Louis, Mo.) solution. The suspended seed was stored at4° C. for 2 days to complete dormancy requirements and ensuresynchronous seed germination (stratification).

Sunshine Mix LP5 (Sun Gro Horticulture, Bellevue, Wash.) was coveredwith fine vermiculite and sub-irrigated with Hoagland's solution untilwet. The soil mix was allowed to drain for 24 hours. Stratified seed wassown onto the vermiculite and covered with humidity domes (KORDProducts, Bramalea, Ontario, Canada) for 7 days.

Seeds were germinated and plants were grown in a Conviron (modelsCMP4030 and CMP3244, Controlled Environments Limited, Winnipeg,Manitoba, Canada) under long day conditions (16 hours light/8 hoursdark) at a light intensity of 120-150 μmol/m² sec under constanttemperature (22° C.) and humidity (40-50%). Plants were initiallywatered with Hoagland's solution and subsequently with deionized waterto keep the soil moist but not wet.

6.2—Agrobacterium Transformation.

An LB+agar plate with erythromycin (Sigma Chemical Co., St. Louis, Mo.)(200 mg/L) or spectinomycin (100 mg/L) containing a streaked DH5α colonywas used to provide a colony to inoculate 4 ml mini prep cultures(liquid LB+erythromycin). The cultures were incubated overnight at 37°C. with constant agitation. Qiagen (Valencia, Calif.) Spin Mini Preps,performed per manufacturer's instructions, were used to purify theplasmid DNA.

Electro-competent Agrobacterium tumefaciens (strains Z707s, EHA101s, andLBA4404s) cells were prepared using a protocol from Weigel andGlazebrook (2002). The competent Agrobacterium cells were transformedusing an electroporation method adapted from Weigel and Glazebrook(2002). 50 μl of competent agro cells were thawed on ice and 10-25 ng ofthe desired plasmid was added to the cells. The DNA and cell mix wasadded to pre-chilled electroporation cuvettes (2 mm). An EppendorfElectroporator 2510 was used for the transformation with the followingconditions, Voltage: 2.4 kV, Pulse length: 5 msec.

After electroporation, 1 ml of YEP broth (per liter: 10 g yeast extract,10 g Bacto-peptone, 5 g NaCl) was added to the cuvette, and the cell-YEPsuspension was transferred to a 15 ml culture tube. The cells wereincubated at 28° C. in a water bath with constant agitation for 4 hours.After incubation, the culture was plated on YEP+agar with erythromycin(200 mg/L) or spectinomycin (100 mg/L) and streptomycin (Sigma ChemicalCo., St. Louis, Mo.) (250 mg/L). The plates were incubated for 2-4 daysat 28° C.

Colonies were selected and streaked onto fresh YEP+agar witherythromycin (200 mg/L) or spectinomycin (100 mg/L) and streptomycin(250 mg/L) plates and incubated at 28° C. for 1-3 days. Colonies wereselected for PCR analysis to verify the presence of the gene insert byusing vector specific primers. Qiagen Spin Mini Preps, performed permanufacturer's instructions, were used to purify the plasmid DNA fromselected Agrobacterium colonies with the following exception: 4 mlaliquots of a 15 ml overnight mini prep culture (liquid YEP+erythromycin(200 mg/L) or spectinomycin (100 mg/L)) and streptomycin (250 mg/L))were used for the DNA purification. An alternative to using Qiagen SpinMini Prep DNA was lysing the transformed Agrobacterium cells, suspendedin 10 μl of water, at 100° C. for 5 minutes. Plasmid DNA from the binaryvector used in the Agrobacterium transformation was included as acontrol. The PCR reaction was completed using Taq DNA polymerase fromTakara Mirus Bio Inc. (Madison, Wis.) per manufacturer's instructions at0.5× concentrations. PCR reactions were carried out in a MJ ResearchPeltier Thermal Cycler programmed with the following conditions; 1) 94°C. for 3 minutes, 2) 94° C. for 45 seconds, 3) 55° C. for 30 seconds, 4)72° C. for 1 minute, for 29 cycles then 1 cycle of 72° C. for 10minutes. The reaction was maintained at 4° C. after cycling. Theamplification was analyzed by 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. A colony was selected whose PCRproduct was identical to the plasmid control.

6.3—Arabidopsis Transformation.

Arabidopsis was transformed using the floral dip method. The selectedcolony was used to inoculate one or more 15-30 ml pre-cultures of YEPbroth containing erythromycin (200 mg/L) or spectinomycin (100 mg/L) andstreptomycin (250 mg/L). The culture(s) was incubated overnight at 28°C. with constant agitation at 220 rpm. Each pre-culture was used toinoculate two 500 ml cultures of YEP broth containing erythromycin (200mg/L) or spectinomycin (100 mg/L) and streptomycin (250 mg/L) and thecultures were incubated overnight at 28° C. with constant agitation. Thecells were then pelleted at approx. 8700× g for 10 minutes at roomtemperature, and the resulting supernatant discarded. The cell pelletwas gently resuspended in 500 ml infiltration media containing: 1/2×Murashige and Skoog salts/Gamborg's B5 vitamins, 10% (w/v) sucrose,0.044 μM benzylamino purine (10 μl/liter of 1 mg/ml stock in DMSO) and300 μl/liter Silwet L-77. Plants approximately 1 month old were dippedinto the media for 15 seconds, being sure to submerge the newestinflorescence. The plants were then laid down on their sides and covered(transparent or opaque) for 24 hours, then washed with water, and placedupright. The plants were grown at 22° C., with a 16-hour light/8-hourdark photoperiod. Approximately 4 weeks after dipping, the seeds wereharvested.

6.4—Selection of Transformed Plants.

Freshly harvested T₁ seed (transformed with native [AAD-1 (v2)] or plantoptimized [AAD-1 (v3)] gene) was allowed to dry for 7 days at roomtemperature. T₁ seed was sown in 26.5×51-cm germination trays (T.O.Plastics Inc., Clearwater, Minn.), each receiving a 200 mg aliquots ofstratified T₁ seed (˜10,000 seed) that had previously been suspended in40 ml of 0.1% agarose solution and stored at 4° C. for 2 days tocomplete dormancy requirements and ensure synchronous seed germination.

Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) wascovered with fine vermiculite and subirrigated with Hoagland's solutionuntil wet, then allowed to gravity drain. Each 40 ml aliquot ofstratified seed was sown evenly onto the vermiculite with a pipette andcovered with humidity domes (KORD Products, Bramalea, Ontario, Canada)for 4-5 days. Domes were removed 1 day prior to initial transformantselection using glufosinate postemergence spray (selecting for theco-transformed PAT gene).

Five to six days after planting (DAP) and again 10 DAP, T₁ plants(cotyledon and 2-4-1f stage, respectively) were sprayed with a 0.2%solution of Liberty herbicide (200 g ai/L glufosinate, Bayer CropSciences, Kansas City, Mo.) at a spray volume of 10 ml/tray (703 L/ha)using a DeVilbiss compressed air spray tip to deliver an effective rateof 280 g ai/ha glufosinate per application. Survivors (plants activelygrowing) were identified 5-7 days after the final spraying andtransplanted individually into 3-inch pots prepared with potting media(Metro Mix 360). Transplanted plants were covered with humidity domesfor 3-4 days and placed in a 22° C. growth chamber as before. Domes weresubsequently removed and plants moved to the greenhouse (22±5° C.,50±30% RH, 14 h light:10 dark, minimum 500 ρE/m²s¹ natural+supplementallight) at least 1 day prior to testing for the ability of AAD-1 (v3)(plant optimized gene) or AAD-1 (v2) (native microbial gene) to providephenoxy auxin herbicide resistance.

Random individual T₁ plants selected for glufosinate resistance abovewere confirmed for expression of the PAT protein using a PAT ELISA kit(Part no. 7000045, Strategic Diagnostics, Inc., Newark, Del.) tonon-destructively confirm fidelity of selection process (manufacturer'sprotocol). Plants were then randomly assigned to various rates ofphenoxy herbicides (dichlorprop or 2,4-D). Phenoxy rates initiallyapplied were 12.5 g ae/ha 2,4-D and 50 or 200 g ae/ha dichlorprop. GR₉₉for Arabidopsis is about 50 g ae/ha 2,4-D and 200 g ae/ha dichlorprop.Elevated rates were applied in subsequent trials (50, 200, 800, or 3200g ae/ha).

All auxin herbicide applications were made using the DeVilbiss sprayeras described above to apply 703 L/ha spray volume (0.4 mlsolution/3-inch pot) or applied by track sprayer in a 187 L/ha sprayvolume. 2,4-D used was either technical grade (Sigma, St. Louis, Mo.)dissolved in DMSO and diluted in water (<1% DMSO final concentration) orthe commercial dimethylamine salt formulation (456 g ae/L, NuFarm, StJoseph, Mo.). Dichlorprop used was technical grade (Sigma, St. Louis,Mo.) dissolved in DMSO and diluted in water (<1% DMSO finalconcentration). As herbicide rates increased beyond 800 g ae/ha, the pHof the spray solution became exceedingly acidic, burning the leaves ofyoung, tender Arabidopsis plants and complicating evaluation of theprimary effects of the herbicides. It became standard practice to applythese high rates of phenoxy herbicides in 200 mM Tris buffer (pH 9.0) toa final pH of ˜7-8.

Some T₁ individuals were subjected to alternative commercial herbicidesinstead of a phenoxy auxin. One point of interest was determiningwhether haloxyfop could be effectively degraded in planta.

Although Arabidopsis, being a dicot, is not an optimal system fortesting ACCase-inhibiting AOPP grass herbicides, AAD-1 (v3)-transformedT₁ plants were subjected to elevated rates (400-1600 g ae/ha) ofRS-haloxyfop acid (internally synthesized) that do cause growthabnormalities and death of wildtype Arabidopsis using the DeVilbisssprayer as described above Injury ratings were taken 7 and 14 days aftertreatment. Likewise, T₁ individuals were treated with thepyridyloxyacetate auxin herbicide, fluroxypyr.

6.5—Results of Selection of Transformed Plants.

The first Arabidopsis transformations were conducted using AAD-1 (v3)(plant optimized gene). T₁ transformants were first selected from thebackground of untransformed seed using a glufosinate selection scheme.Over 400,000 T₁ seed were screened and 493 glufosinate resistant plantswere identified (PAT gene), equating to a transformation/selectionfrequency of 0.12%. Depending on the lot of seed tested, this rangedfrom 0.05-0.23% (see Table 15). A small lot of AAD-1 (v2)(native)-transformed seed were also selected using the glufosinateselection agent. Two hundred seventy eight glufosinate-resistant T₁individuals were identified out of 84,000 seed screened (0.33%transformation/selection frequency).

TABLE 15 Selection of AAD-1 (v3) (plant optimized), or AAD-1 (v2)(native), AAD-2 (v1) (native), or plant optimized AAD-2 (v2)-transformedT₁ individual plants using glufosinate and 2,4-D. Total seed Selection %of selected Selection Codon sown and Number of Selection rate plantsexpressing agent Gene bias screened resistant T₁ rate range PAT³Glufosinate¹ AAD-1 (v2) n 84,000 278 0.33% 0.33% nd⁴ Glufosinate¹ AAD-1(v3) p 400,500 493 0.12% 0.05 to 0.23% 97% 2,4-D² AAD-1(v3) p 70,000 530.08% 0.07 to 0.08% 96% Glufosinate¹ AAD-2 (v1) n 1,301,500 228 0.018%0.007 to 0.021% 100%  Glufosinate¹ AAD-2(v2) p 200,000 224 0.11% 0.11%nd⁴ ¹Glufosinate selection scheme: 280 g ai/ha glufosinate applied 5-6 +10 DAP ²2,4-D selection scheme: 50 g ai/ha 2,4-D applied 5-7 + 10-14 DAP³PAT protein expression determined by PAT ELISA strips ⁴nd, notdetermined ⁵codon bias, n-native microbial gene, p = plant optimized

T₁ plants selected above were subsequently transplanted to individualpots and sprayed with various rates of commercial aryloxyalkanoateherbicides. Table 16 compares the response of native AAD-1 (v2) andplant optimized AAD-1 (v3) genes to impart 2,4-D resistance toArabidopsis T₁ transformants Both genes imparted resistance toindividual T₁ Arabidopsis plants. Within a given treatment, the level ofplant response varied greatly and can be attributed to the fact eachplant represents an independent transformation event. Of important note,at each 2,4-D rate tested, there were individuals that were unaffectedwhile some were severely affected. An overall population injury averageby rate is presented in Table 16 simply to demonstrate the significantdifference between the plants transformed with AAD-1 (v2) or AAD-1 (v3)versus the wildtype or PAT/Cry1F transformed controls. Also evident isthat tolerance appears to be significantly greater (frequency andoverall level of individual response) for the plant optimized sequenceAAD-1 (v3) versus the native sequence AAD-1 (v2) (see Table 16). Higherrates of 2,4-D (up to 3,200 g ae/ha) have been applied to additional T₁individuals expressing AAD-1 (v3). Injury levels tend to be greater andthe frequency of highly resistant plants is lower at these elevatedrates (6× field rates). Also at these high rates, the spray solutionbecomes highly acidic unless buffered. Arabidopsis grown mostly in thegrowth chamber has a very thin cuticle and severe burning effects cancomplicate testing at these elevated rates. Nonetheless, someindividuals have survived 3,200 g ae/ha 2,4-D with little or no injury.

Table 16. AAD-1 v3 (plant optimized), or AAD-1 v2 (native), or AAD-2(native)-transformed T₁ Arabidopsis response to a range of 2,4-D ratesapplied postemergence. Response is presented in terms of % visual injury2 WAT. Data are presented as a histogram of individuals exhibitinglittle or no injury (<20%), moderate injury (20-40%), or severe injury(>40%). Since each T₁ is an independent transformation event, one canexpect significant variation of individual T₁ responses within a givenrate. An arithmetic mean and standard deviation is presented for eachtreatment. The range in individual response is also indicated in thelast column for each rate and transformation. PAT/Cry1F-transformedArabidopsis served as an auxin-sensitive transformed control. WildtypeArabidopsis is untransformed.

TABLE 16 AAD-1 v3 (plant optimized), or AAD-1 v2 (native), or AAD-2(native)-transformed T₁ Arabidopsis response to a range of 2,4-D ratesapplied postemergence. % Injury % Injury Std Averages <20% 20-40% >40%Ave Dev Native AAD-1 (v2) gene Untreated control-buffer 20 6 7 25.3 34.750 g ae/ha 2,4-D 55 16 9 14.8 22.7 200 g ae/ha 2,4-D 45 11 24 34.1 39.3800 g ae/ha 2,4-D 11 32 37 52.5 34.2 Native AAD-2 gene Untreatedcontrol-buffer 4 1 1 25.0 21.7 50 g ae/ha 2,4-D 1 2 11 68.2 30.2 200 gae/ha 2,4-D 0 3 11 82.7 28.8 800 g ae/ha 2,4-D 0 0 14 99.8 0.8 RebuiltAAD-1 (v3) gene Untreated control-buffer 9 0 0 0.0 0.0 50 g ae/ha 2,4-D10 1 5 24.3 35.9 200 g ae/ha 2,4-D 11 4 1 14.0 25.9 800 g ae/ha 2,4-D 114 1 14.7 26.1 Wildtype Untreated control-buffer 11 0 0 0.0 0.0 50 gae/ha 2,4-D 0 0 15 90.0 0.0 200 g ae/ha 2,4-D 0 0 15 95.1 0.5 800 gae/ha 2,4-D 0 0 15 100.0 0.0 PAT/Cry1F (transformed control) Untreatedcontrol-buffer 11 0 0 0.0 0.0 50 g ae/ha 2,4-D 0 0 15 90.7 4.2 200 gae/ha 2,4-D 0 0 15 97.2 1.7 800 g ae/ha 2,4-D 0 0 15 100.0 0.0

Table 17 shows a similar dose response of T₁ Arabidopsis to thephenoxypropionic acid, dichlorprop. Similar trends were seen as with2,4-D, indicating the chiral propionic side chain indeed serves as anacceptable substrate. Next, it was determined that a degree of increasedhaloxyfop tolerance could be imparted to transformed Arabidopsis atelevated rates of 400-1,600 g ae/ha (Table 18). Normal field use ratefor haloxyfop (a grass-specific herbicide) is around 50-70 g ae/ha.Dicots are generally considered naturally tolerant to AOPP herbicides;however, severe physiological effects do occur in Arabidopsis at theseelevated rates. Some AAD1 (v3) transformed individuals did exhibitincreased tolerance to haloxyfop. This provides the first in planta datathat AAD-1 (v3) will provide AOPP resistance. No resistance was observedwith fluroxypyr (a pryridyloxyacetate auxin) in transformed Arabidopsis,consistent with in vitro work using heterologously expressed enzyme.

TABLE 17 T₁ Arabidopsis response to a range of dichlroprop rates appliedpostemergence. Response is presented in terms of % visual injury 2 WAT.Data are presented as a histogram of individuals exhibiting little or noinjury (<20%), moderate injury (20-40%), or severe injury (>40%). Sinceeach T₁ is an independent transformation event, one can expectsignificant variation of individual T₁ responses within a given rate. Anarithmetic mean and standard deviation is presented for each treatment.The range in individual response is also indicated in the last columnfor each rate and transformation. PAT/Cry1F-transformed Arabidopsisserved as an auxin-sensitive transformed control. Wildtype Arabidopsisis untransformed. Table 17. T₁ Arabidopsis response to a range ofdichlroprop rates applied postemergence. % Injury % Injury Averages <20%20-40% >40% Ave Std Dev Range AAD-1 v3 Untreated control 3 0 0 0.0 0.0 012.5 g ae/ha RS-dichlorprop 7 1 0 5.0 7.6 0-20 50 g ae/ha RS-dichlorprop7 1 0 3.1 8.8 0-25 200 g ae/ha RS-dichlorprop 4 1 3 40.0 50.1  0-100 800g ae/ha RS-dichlorprop 0 5 3 51.9 40.0 20-100 PAT/Cry1F Untreatedcontrol 3 0 0 0.0 0.0 0 12.5 g ae/ha RS-dichlorprop 0 6 2 38.1 25.320-95  50 g ae/ha RS-dichlorprop 0 0 8 80.0 25.3 50-100 200 g ae/haRS-dichlorprop 0 0 8 98.3 2.2 95-100 800 g ae/ha RS-dichlorprop 0 0 8100.0 0.0 100  Wildtype Untreated control 3 0 0 0.0 0.0 0 12.5 g ae/haRS-dichlorprop 3 0 0 13.3 2.9 10-15  50 g ae/ha RS-dichlorprop 0 0 353.3 5.8 50-60  200 g ae/ha RS-dichlorprop 0 0 3 95.0 5.0 90-100 800 gae/ha RS-dichlorprop 0 0 3 100.0 0.0 100 

TABLE 18 T1 Arabidopsis response to a range of haloxyfop rates appliedpostemergence at artificially high rates attempting to show tolerance ofthe dicot Arabidopsis to the graminicide. Response is presented in termsof % visual injury 2 WAT. Data are presented as a histogram ofindividuals exhibiting little or no injury (<20%), moderate injury(20-40%), or severe injury (>40%). Since each T1 is an independenttransformation event, one can expect significant variation of individualT1 responses within a given rate. An arithmetic mean and standarddeviation is presented for each treatment. The range in individualresponse is also indicated in the last column for each rate andtransformation. PAT/Cry1F-transformed Arabidopsis served as anauxin-sensitive transformed control. Wildtype Arabidopsis isuntransformed. Table 18. T₁ Arabidopsis response to a range of haloxyfoprates applied postemergence at artificially high rates attempting toshow tolerance of the dicot Arabidopsis to the graminicide. % Injury %Injury Averages <20% 20-40% >40% Ave Std Dev Range AAD-1 v3 Untreatedcontrol 3 0 0 0.0 0.0 0 100 g ae/ha haloxyfop 4 0 0 0.0 0.0 0 200 gae/ha haloxyfop 4 0 0 0.0 0.0 0 400 g ae/ha haloxyfop 3 1 0 6.3 9.5 0-20800 g ae/ha haloxyfop 1 1 2 46.3 42.7 0-85 1600 g ae/ha haloxyfop 1 0 365.0 47.3  0-100 PAT/Cry1F Untreated control 3 0 0 0.0 0.0 0 100 g ae/hahaloxyfop 4 0 0 0.0 0.0 0 200 g ae/ha haloxyfop 4 0 0 10.0 0.0 10  400 gae/ha haloxyfop 0 4 0 27.5 5.0 20-30  800 g ae/ha haloxyfop 0 0 4 78.86.3 70-85  1600 g ae/ha haloxyfop 0 0 4 47.5 43.5 80-100 WildtypeUntreated control 3 0 0 0.0 0.0 0 100 g ae/ha haloxyfop 3 0 0 0.0 0.0 0200 g ae/ha haloxyfop 3 0 0 0.0 0.0 0 400 g ae/ha haloxyfop 0 3 0 20.00.0 20  800 g ae/ha haloxyfop 0 0 3 73.3 10.4 70-85  1600 g ae/hahaloxyfop 0 0 3 93.3 11.5 80-100

6.6—AAD-1 (v3) as a Selectable Marker.

The ability to use AAD-1 (v3) as a selectable marker using 2,4-D as theselection agent was analyzed initially with Arabidopsis transformed withas described above. T₁ seed transformed with PAT and AAD1 (v3) (pDAB721) were sown into flats and germinated as described above and comparedto similar seed treated with the normal glufosinate selection scheme (5and 10 DAP). 2,4-D (50 g ae/ha) was applied to seedling Arabidopsis aspreviously done with glufosinate. Variation in number of applicationsand timing of application were tested. Each tray of plants receivedeither one or two application timings of 2,4-D in one of the followingtreatment schemes: 5+10 DAP, 5+14 DAP, 10 DAP, 10+14 DAP, 14 DAP. Plantswere identified as Resistant or Sensitive 19 DAP and ELISA test stripsrun to determine frequency of successfully co-transforming an active PATgene.

Fifty-three out of 70,000 seed planted were identified as resistant to2,4-D. ELISA was used to screen a subset of 44-individuals from thispopulation for PAT protein expression. Ninety six percent of theindividuals were positive indicating the presence of the co-transformedgene, PAT. The low number of negative ELISA results (4%) is in line witha 3% error rate in populations of glufosinate-resistant plants (Table15). The efficiency of selection appears to be somewhat less with 2,4-D(0.08%) vs. glufosinate (0.12%); however, the range of selection ratesacross all experiments would indicate both selection agents are equallygood for selecting Arabidopsis transformed with AAD-1 (v3) or PAT genes,respectively. Two successive applications most accurately identifyresistant individuals with both herbicides tested.

6.7—Heritability.

A variety of T₁ events were self-pollinated to produce T₂ seed. Theseseed were progeny tested by applying 2,4-D (200 g ae/ha) to 100 randomT₂ siblings. Each individual T₂ plant was transplanted to 7.5-cm squarepots prior to spray application (track sprayer at 187 L/ha applicationsrate). More than 60% of the T₁ families (T₂ plants) segregated in theanticipated 3 Resistant:1 Sensitive model for a dominantly inheritedsingle locus with Mendelian inheritance as determined by Chi squareanalysis (P>0.05).

Seed were collected from 12 to 20 T₂ individuals (T₃ seed). Twenty-fiveT₃ siblings from each of eight randomly-selected T₂ families wereprogeny tested as previously described. Approximately one-third of theT₂ families anticipated to be homozygous (non-segregating populations)have been identified in each line tested: ranging in frequency from oneto four out of the eight families tested. These data show AAD1 (v3) isstably integrated and inherited in a Mendelian fashion to at least threegenerations.

6.8—Additional Herbicide Resistance Attributable to AAD-1 inArabidopsis.

The ability of AAD-1 (v3) to provide resistance to otheraryloxyphenoxyalkanoate herbicides in transgenic Arabidopsis wasdetermined using a modified in vitro plate assay. Seeds from wild-typeArabidopsis thaliana as well as Arabidopsis thaliana containing theplant optimized AAD-1 (v3) gene (T₄ homozygous plants id=PAAD1.315.064)were sterilized by agitating for 10 min in a 50% bleach solution. Theseseeds were then rinsed four times with sterile water to remove thebleach.

Dose response assays utilized a nutrient media (see below) supplementedwith various rates of test compounds. The test compounds were added tothe heated media (55° C.) as concentrated solutions in DMSO. Controlwells had the appropriate amount of DMSO without any additionalcompound. The final concentration of DMSO never exceeded 1% (v/v). Afterthorough mixing, a 6 mL aliquot of the warm media containing theappropriate concentration of compound was added to each well of a6-well, flat bottom, polystyrene tissue culture tray (Falcon 353046,Becton Dickson and Company, Franklin Lakes, N.J.). After the mediasolidified, approximately 20 to 30 Arabidopsis seeds were applied on topof the solidified media and the remaining 2 mL of media was poured overthe seeds. The plates were lightly agitated to disperse the seeds,covered and allowed to cool until the media had completely solidified.The plates were incubated for 7 days at 25° C. under continualfluorescent lighting (75 μE m⁻² s⁻¹). Nutrient Media Composition was asdescribed in Example 2.2 and in Somerville and Orgen (1982).

Assessment of growth reduction. The apical portion of the Arabidopsisplants grown in the treated media was assessed visually relative to theapical portion of the plants grown in the media containing only DMSO.Values were recorded as % growth reduction. Assessments of root growthinhibition of the Arabidopsis plants grown in the treated media wereachieved by carefully extracting the plants from the media and measuringthe length of the root. These root lengths were then compared to theroot length of the control plants to determine a % growth reduction. Aminimum of five plants were assessed for each treatment. The valuesrecorded are an average of all the plants assessed. The calculatedconcentration to reach 50% inhibition effect (I₅₀) were determined forboth root and shoots of wildtype and AAD-1-transformed Arabidopsis. Theratios of resistant to sensitive biotypes are included in Table 19. Aratio >2 for both root and shoot measurements generally signifiessignificant resistance. The higher the ratio, the greater the level ofresistance. All commercial phenoxy auxins showed significant levels ofresistance including oxyacetic acids (2,4-D and MCPA) as well asoxypropionic acids (dichlorprop and mecoprop). In fact, the chronic rootassessment shows resistance to the oxypropionic acid is higher withAAD-1 (v3) than for the oxyacetic acids, consistent with enzymaticcharacteristics of AAD-1 (v1). Assessment of other auxins containingpyridine rings showed AAD-1 (v3) did not effectively protect Arabidopsisform pyridyloxyacetates herbicides, triclopyr and fluroxypyr, or thepicolinic acid herbicide, picloram. The broad phenoxy auxin resistanceis the first reported in planta. The alternative auxins to which AAD-1does not protect would be viable tools for the control and containmentof AAD-1-transformed commercial crops or experimental plant species.

TABLE 19 In vitro plate test assessment of herbicide substrate crossresistance afforded by AAD-1 (v3) in homozygous T₄ Arabidopsis (ARBTH).wt ARBTH PAAD1.315.064 wt shoot T3 ARBTH ARBTH PAAD1.315.064 I50 shootI50 root I50 ARBTH root 150 shoot root Compound Structure ppm ratioratio Phenoxy auxins 2,4-D

0.2 10 <0.01 0.04 50 >4 dichlorprop

2 5 0.01 1.5 2.5 150 Mecoprop

2 25 0.01 1.5 12.5 150 MCPA

0.2 1 0.01 0.03 5 3 2,4,5-T

1.5 10 <0.01 <0.01 6.67 NA Pyridine auxins Fluroxypyr

2 1 0.2 0.2 0.5 1 Triclopyr

0.2 0.04 0.02 0.02 0.2 1 Picloram

1 0.5 0.3 0.15 0.5 0.5

6.9—Foliar Applications Herbicide Resistance in AAD-1 Arabidopsis.

The ability of AAD-1 (v3) to provide resistance to otheraryloxyphenoxyalkanoate auxin herbicides in transgenic Arabidopsis wasdetermined by foliar application of various substrates described inExample 6.8. T₄ generation Arabidopsis seed, homozygous for AAD-1 (v3)(line AAD1.01.315.076) was stratified, and sown into selection traysmuch like that of Arabidopsis (Example 6.4). A transformed-control linecontaining PAT and the insect resistance gene Cry1F was planted in asimilar manner Seedlings were transferred to individual 3-inch pots inthe greenhouse. All plants were sprayed with the use of a track sprayerset at 187 L/ha. The plants were sprayed from a range of phenoxy auxinherbicides: 12.5-1600 g ae/ha 2,4-D dimethylamine salt (DMA) (RiversideChemicals), 12.5-1600 g ae/ha mecoprop (AH Marks), 50-3200 g ae/haR-dichlorprop (AH Marks), 8.75-1120 g ae/ha 2,4,5-T (technical grade);pyridyloxyacetates herbicides 50-3200 g ae/ha triclopyr (DowAgroSciences) and 50-3200 g ae/ha fluroxypyr (Dow AgroSciences); and the2,4-D metabolite resulting from AAD-1 activity, 2,4-dichlorophenol (DCP,Sigma) (at 50-3200 g ae/ha, technical grade). All applications wereformulated in 200 mM Hepes buffer (pH 7.5). Each treatment wasreplicated 3-4 times. Plants were evaluated at 3 and 14 days aftertreatment and are averaged over two experiments.

These results (see Table 20) confirm that AAD-1 (v3) in Arabidopsisprovides robust resistance to the phenoxyacetic auxins, phenoxypropionicauxins, but have not shown significant cross resistance to thepyridyloxyacetic auxins tested and corroborates the in vitro enzyme andwhole plate substrate specificity data. Additionally, there is no effectof the metabolite, 2,4-dichlorphohenol (DCP), on wildtype or transgenicArabidopsis.

TABLE 20 Comparison of homozygous T₄ AAD-1 (v3) and wildtype Arabidopsisplant response to various foliar-applied auxinic herbicides. Ave %Injury 14 DAT AAD1.01.315.076.T₄ PatCry1f - Herbicide Treatmenthomozygous AAD1 plants Control Phenoxypropionic auxins 50 g ae/haR-Dichlorprop 3 31 200 g ae/ha R-Dichlorprop 3 73 800 g ae/haR-Dichlorprop 3 89 3200 g ae/ha R-Dichlorprop 3 95 12.5 g ae/ha Mecoprop3 0 25 g ae/ha Mecoprop 0 2 50 g ae/ha Mecoprop 0 17 100 g ae/haMecoprop 0 33 200 g ae/ha Mecoprop 3 62 400 g ae/ha Mecoprop 0 78 800 gae/ha Mecoprop 0 93 1600 g ae/ha Mecoprop 0 100 Phenoxyacetic auxins12.5 g ae/ha 2,4-D DMA 0 67 25 g ae/ha 2,4-D DMA 0 78 50 g ae/ha 2,4-DDMA 0 93 100 g ae/ha 2,4-D DMA 0 100 200 g ae/ha 2,4-D DMA 0 100 400 gae/ha 2,4-D DMA 0 100 800 g ae/ha 2,4-D DMA 0 100 1600 g ae/ha 2,4-D DMA0 100 8.75 g ae/ha 2,4,5-T 0 0 17.5 g ae/ha 2,4,5-T 3 20 35 g ae/ha2,4,5-T 0 43 70 g ae/ha 2,4,5-T 3 85 140 g ae/ha 2,4,5-T 0 95 280 gae/ha 2,4,5-T 0 98 560 g ae/ha 2,4,5-T 17 100 1120 g ae/ha 2,4,5-T 3 100Pyridyloxyacetic auxins 50 g ae/ha Triclopyr 31 36 200 g ae/ha Triclopyr58 65 800 g ae/ha Triclopyr 74 84 3200 g ae/ha Triclopyr 97 95 50 gae/ha Fluroxypyr 48 76 200 g ae/ha Fluroxypyr 75 85 800 g ae/haFluroxypyr 88 85 3200 g ae/ha Fluroxypyr 95 95 Inactive DCP metabolite50 g ae/ha 2,4-DCP 0 0 200 g ae/ha 2,4-DCP 0 0 800 g ae/ha 2,4-DCP 0 03200 g ae/ha 2,4-DCP 0 0

6.10—Relationship of Plant Growth to AAD-1 (v3) Expression inArabidopsis.

An experiment was designed to examine if the level of AAD-1 (v3)expression in Arabidopsis varies at different growth stages. Ahigh-tolerance, homozygous, AAD-1 (v3) T₄ line (id=PAAD1.01.345.163) wasgrown in greenhouse. Half of the plants were treated with 800 g ae/ha of2,4-D (as previously described) while the other half were not treated.Two leaves, the 3rd leaf from the top and the 5th leaf from the bottom,were harvested from 5 plants, both treated and untreated, and analyzedby ELISA and Western Blotting (as described in Example 11) experimentsat 4, 10, 14, 20 and 25 DAT. FIGS. 8A and 8B showed that there wasstatistically no difference in AAD-1 (v3) expression between young andold leaves. In addition, the herbicide 2,4-D had little impact on theexpression level of AAD-1 (v3) protein. The protein levels accumulatedin older plants with some significant protein degradation at the latertime points.

In a separate experiment, four different homozygous T₄ lines ofArabidopsis displaying different tolerance level to the herbicide 2,4-Dwere sprayed with various levels (0, 200, 800 and 3200 g/ha) of 2,4-Dand their herbicide injury and AAD-1 (v3) expression were examined Fourdays after the herbicide treatment, little injury was observed in threeof the four lines, even at the highest dose tested (FIG. 9A). Theseplants also expressed high level of AAD-1 (v3), from 0.1 to 0.25% of(FIG. 9B). On the contrary, the low tolerance line expressed less than0.1% of AAD-1 (v3) in TSP and suffered observable injury. Moreimportantly, they recovered from the injury on 14 DAT (FIG. 9A),indicating that the low level of AAD-1 (v3) expression was able toprotect the plants from the serious herbicide damage. All control plantssuffered serious injury and died 14 DAT at doses 800 g ae/ha 2,4-D andabove.

6.11—Molecular Analysis of AAD-1 (v3) Arabidopsis.

Invader Assay (methods of Third Wave Agbio Kit Procedures) for PAT genecopy number and/or Southern blot analysis was performed with total DNAobtained from Qiagen DNeasy kit on multiple AAD-1 (v3) homozygous linesto determine stable integration of the plant transformation unitcontaining PAT and AAD-1 (v3). Analysis assumed direct physical linkageof these genes as they were contained on the same plasmid.

For Southern analysis, a total of 1 μg of DNA was subjected to anovernight digest of Nsi I for pDAB721 to obtain integration data. Thesamples were run on a large 0.85% agarose gel overnight at 40 volts. Thegel was then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gelwas then neutralized in 0.5 M Tris HCl, 1.5 M NaCl pH of 7.5 for 30minutes. A gel apparatus containing 20SSC was then set up to obtain agravity gel to nylon membrane (Millipore INYC00010) transfer overnight.After the overnight transfer the membrane was then subjected to UV lightvia a crosslinker (stratagene UV stratalinker 1800) at 1200 X100microjoules. The membrane was then washed in 0.1% SDS, 0.1 SSC for 45minutes. After the 45 minute wash, the membrane was baked for 3 hours at80° C. and then stored at 4° C. until hybridization. The hybridizationtemplate fragment consisted of the prepared primers (Pat 5-3AGATACCCTTGGTTGGTTGC) (SEQ ID NO:23) and (Pat 3-3 CAGATGGATCGTTTGGAAGG)(SEQ ID NO:24) designed to obtain the coding region of PAT. The productwas run on a 1% agarose gel and excised and then gel extracted using theQiagen (28706) gel extraction procedure. The membrane was then subjectedto a pre-hybridization at 60° C. step for 1 hour in Perfect Hyb buffer(Sigma H7033). The Prime it RmT dCTP-labeling rxn (Stratagene 300392)procedure was used to develop the p32 based probe (Perkin Elmer). Theprobe was cleaned up using the Probe Quant. G50 columns (Amersham27-5335-01). Two million counts CPM per ml of Perfect Hyb buffer wasused to hybridize the southern blots overnight. After the overnighthybridization the blots were then subjected to two 20 minute washes at65° C. in 0.1% SDS, 0.1 SSC. The blots were then exposed to filmovernight, incubating at −80° C.

Results showed all 2,4-D resistant plants assayed contained PAT (andthus by inference, AAD-1 (v3)). Copy number analysis showed totalinserts ranged from 1 to >10 copies. This correlates, too, with theAAD-1 (V3) protein expression data indicating that the presence of theenzyme yields significantly high levels of resistance (>>200 fold) toall commercially available phenoxyacetic and phenoxypropionic acids.

6.12—Arabidopsis Transformed with Molecular Stack of AAD-1 (v3) andGlyphosate Resistance Gene.

T₁ Arabidopsis seed was produced, as previously described, containingpDAB3230 plasmid (AAD-1 (v3)+EPSPS) coding for a putative glyphosateresistance trait. T₁ transformants were selected using AAD-1 (v3) as theselectable marker as described in example 6.6, except the 2,4-D rateused was 75 g ae/ha. Twenty-four T₁ individually transformed events wererecovered from the first selection attempt and transferred to three-inchpots in the greenhouse as previously described. Three different controlArabidopsis lines were also tested: wildtype Columbia-0, AAD-1 (v3)+PATT₅ homozygous lines (pDAB721-transformed), and PAT+Cry1F homozygous line(transformed control). Only pDAB3230 plants were pre-selected at theseedling stage for 2,4-D tolerance. Four days after transplanting,plants were evenly divided for foliar treatment by track sprayer aspreviously described with 0, 26.25, 105, 420, or 1680 g ae/ha glyphosate(Glyphomax Plus, Dow AgroSciences) in 200 mM Hepes buffer (pH 7.5). Alltreatments were replicated 4 or 5 times. Plants were evaluated 7 and 14days after treatment. I₅₀ values were calculated and show >14 fold levelof tolerance imparted by EPSPS molecularly stacked with AAD-1 (v3) (seeFIG. 10). AAD-1 (v3) did not provide resistance to glyphosate itself(re: pDAB721 response). These T₁ plants will be grown to seed,self-pollinated to yield T₂ seed. The pDAB 3230 T₁ plants havedemonstrated tolerance to lethal doses of 2,4-D and glyphosate. T₂plants will be further tested to demonstrate these co-transformed plantswill withstand glyphosate+2,4-D treatments applied in tankmix asdescribed in Example 21 and shown for AAD-1 (v3)-transformed corn inExample 8.

Example 7—WHISKERS-Mediated Transformation into Maize, and Use of AAD-1(v3) as a Selectable Marker

7.1—Cloning of AAD-1 (v3).

The AAD-1 (v3) fragment was received on an NcoI/SacI fragment. ConstructpDAB4005 was digested with NcoI and SacI and the 5175 bp backbonefragment isolated. The two fragments were ligated together using T₄ DNAligase and transformed into DH5α cells. Minipreps were performed on theresulting colonies using Qiagen's QIA Spin mini prep kit, and thecolonies were digested to check for orientation. The correctintermediate plasmid was named pDAB3403. Both pDAB3403 and pDAB8505(OsAct1/PAT/ZmLip) were digested with NotI. The 3442 bp band frompDAB3403 and the 11017 bp band from pDAB8505 were isolated and purified.The fragments were ligated together, transformed into DH5a, and theresulting plasmids were screened for orientation. The final constructwas designated pDAB3404, which contains ZmUbi1/po-aad1/ZmPer5::OsAct1/PAT/ZmLip.

7.2—Callus/Suspension Initiation.

To obtain immature embryos for callus culture initiation, F₁ crossesbetween greenhouse-grown Hi-II parents A and B (Armstrong et al. 1991)were performed. When embryos were 1.0-1.2 mm in size (approximately 9-10days post-pollination), ears were harvested and surface sterilized byscrubbing with Liqui-Nox® soap, immersed in 70% ethanol for 2-3 minutes,then immersed in 20% commercial bleach (0.1% sodium hypochlorite) for 30minutes.

Ears were rinsed in sterile, distilled water, and immature zygoticembryos were aseptically excised and cultured on 15Ag10 medium (N6Medium (Chu et al., 1975), 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/Lcasein hydrolysate (enzymatic digest), 25 mM L-proline, 10 mg/L AgNO₃,2.5 g/L Gelrite, pH 5.8) for 2-3 weeks with the scutellum facing awayfrom the medium. Tissue showing the proper morphology (Welter et al.,1995) was selectively transferred at biweekly intervals onto fresh15Ag10 medium for about 6 weeks, then transferred to 4 medium (N6Medium, 1.0 mg/L 2,4-D, 20 g/L sucrose, 100 mg/L casein hydrolysate(enzymatic digest), 6 mM L-proline, 2.5 g/L Gelrite, pH 5.8) atbi-weekly intervals for approximately 2 months.

To initiate embryogenic suspension cultures, approximately 3 ml packedcell volume (PCV) of callus tissue originating from a single embryo wasadded to approximately 30 ml of H9CP+ liquid medium (MS basal saltmixture (Murashige and Skoog, 1962), modified MS Vitamins containing10-fold less nicotinic acid and 5-fold higher thiamine-HCl, 2.0 mg/L2,4-D, 2.0 mg/L α-naphthaleneacetic acid (NAA), 30 g/L sucrose, 200 mg/Lcasein hydrolysate (acid digest), 100 mg/L myo-inositol, 6 mM L-proline,5% v/v coconut water (added just before subculture), pH 6.0). Suspensioncultures were maintained under dark conditions in 125 ml Erlenmeyerflasks in a temperature-controlled shaker set at 125 rpm at 28° C. Celllines typically became established within 2 to 3 months afterinitiation. During establishment, suspensions were subcultured every 3.5days by adding 3 ml PCV of cells and 7 ml of conditioned medium to 20 mlof fresh H9CP+ liquid medium using a wide-bore pipette. Once the tissuestarted doubling in growth, suspensions were scaled-up and maintained in500 ml flasks whereby 12 ml PCV of cells and 28 ml conditioned mediumwas transferred into 80 ml H9CP+ medium. Once the suspensions were fullyestablished, they were cryopreserved for future use.

7.3—Cryopreservation and Thawing of Suspensions.

Two days post-subculture, 4 ml PCV of suspension cells and 4 ml ofconditioned medium were added to 8 ml of cryoprotectant (dissolved inH9CP+ medium without coconut water, 1 M glycerol, 1 M DMSO, 2 M sucrose,filter sterilized) and allowed to shake at 125 rpm at 4° C. for 1 hourin a 125 ml flask. After 1 hour 4.5 ml was added to a chilled 5.0 mlCorning cryo vial. Once filled individual vials were held for 15 minutesat 4° C. in a controlled rate freezer, then allowed to freeze at a rateof −0.5° C./minute until reaching a final temperature of −40° C. Afterreaching the final temperature, vials were transferred to boxes withinracks inside a Cryoplus 4 storage unit (Forma Scientific) filled withliquid nitrogen vapors.

For thawing, vials were removed from the storage unit and placed in aclosed dry ice container, then plunged into a water bath held at 40-45°C. until “boiling” subsided. When thawed, contents were poured over astack of ˜8 sterile 70 mm Whatman filter papers (No. 4) in covered100×25 mm Petri dishes. Liquid was allowed to absorb into the filtersfor several minutes, then the top filter containing the cells wastransferred onto GN6 medium (N6 medium, 2.0 mg/L 2,4-D, 30 g/L sucrose,2.5 g/L Gelrite, pH 5.8) for 1 week. After 1 week, only tissue withpromising morphology was transferred off the filter paper directly ontofresh GN6 medium. This tissue was subcultured every 7-14 days until 1 to3 grams was available for suspension initiation into approximately 30 mLH9CP+ medium in 125 ml Erlenmeyer flasks. Three milliliters PCV wassubcultured into fresh H9CP+ medium every 3.5 days until a total of 12ml PCV was obtained, at which point subculture took place as describedpreviously.

7.4—Dose Response of Non-Transformed Tissue to Haloxyfop Acid.

Non-transformed donor Hi-II cell lines were pooled together,WHISKERS-treated without DNA, filtered onto GN6 medium at the rate of 6ml per filter, and allowed to callus for 2-3 weeks at 28° C.Approximately 200 mg of callused suspension tissue was transferred pertreatment to selection media in 60×20 mm plates containing 30, 100 or300 nM R-haloxyfop acid. Three replicates were used per concentration. Acontrol medium containing 1 mg/L bialaphos (from Herbiace commercialformulation, Meiji Seika, Japan), GN6 (1H), was also included forcomparison. Callus was removed after 2 weeks, weighed, and transferredto fresh media of the same concentration for another 2 weeks. After atotal of 4 weeks elapsed time, tissue was removed, weighed a final time,and then discarded. Results are shown in FIG. 11.

Two separate dose response studies of callus tissue to the phenoldegradation products of haloxyfop and cyhalofop, respectively, were alsocompleted to confirm that this end product would not be deleterious tocallus growth. Data from a cyhalofop phenol dose response (see FIG. 12)shows that at 1 μM cyhalofop phenol, growth is still 76% as high as thecontrol without cyhalofop phenol. Data from a haloxyfop phenol doseresponse showed that even at 300 nM haloxyfop phenol, growth was equalto or greater than the control lacking haloxyfop phenol (data notshown).

7.5—WHISKERS-Mediated Transformation Using Bialaphos Selection.

Approximately 24 hours prior to transformation, 12 ml PCV of previouslycryopreserved embryogenic maize suspension cells plus 28 ml ofconditioned medium was subcultured into 80 ml of GN6 liquid medium (GN6medium lacking Gelrite) in a 500 ml Erlenmeyer flask, and placed on ashaker at 125 rpm at 28° C. This was repeated 2 times using the samecell line such that a total of 36 ml PCV was distributed across 3flasks. After 24 hours the GN6 liquid media was removed and replacedwith 72 ml GN6 S/M osmotic medium (N6 Medium, 2.0 mg/L 2,4-D, 30 g/Lsucrose, 45.5 g/L sorbitol, 45.5 g/L mannitol, 100 mg/L myo-inositol, pH6.0) per flask in order to plasmolyze the cells. The flasks were placedon a shaker in the dark for 30-35 minutes, and during this time a 50mg/ml suspension of silicon carbide whiskers was prepared by adding theappropriate volume of GN6 S/M liquid medium to ˜405 mg ofpre-autoclaved, silicon carbide whiskers (Advanced Composite Materials,Inc.).

After incubation in GN6 S/M, the contents of each flask were pooled intoa 250 ml centrifuge bottle. Once all cells settled to the bottom, allbut ˜14 ml of GN6 S/M liquid was drawn off and collected in a sterile1-L flask for future use. The pre-wetted suspension of whiskers wasvortexed for 60 seconds on maximum speed and 8.1 ml was added to thebottle, to which 170 μg DNA was added as a last step. The bottle wasimmediately placed in a modified Red Devil 5400 commercial paint mixerand agitated for 10 seconds. After agitation, the cocktail of cells,media, whiskers and DNA was added to the contents of the 1-L flask alongwith 125 ml fresh GN6 liquid medium to reduce the osmoticant. The cellswere allowed to recover on a shaker for 2 hours before being filteredonto Whatman #4 filter paper (5.5 cm) using a glass cell collector unitthat was connected to a house vacuum line.

Either 3 or 6 mL of dispersed suspension was pipetted onto the surfaceof the filter as the vacuum was drawn. Filters were placed onto 60×20 mmplates of GN6 medium. Plates were cultured for 1 week at 28° C. in adark box that was loosely sealed with a single layer of plastic (<2 milsthick) to minimize evaporation of the individual plates.

After 1 week, filter papers were transferred to 60×20 mm plates of GN6(1H) medium (N6 Medium, 2.0 mg/L 2,4-D, 30 g/L sucrose, 100 mg/Lmyo-inositol, 1.0 mg/L bialaphos, 2.5 g/L Gelrite, pH 5.8) or GN6D (1H)medium (same as GN6 (1H) except with 8.0 mg/L dicamba and 0.8 mg/L2,4-D).

Plates were placed in boxes and cultured as before for an additionalweek. Two weeks post-transformation, the tissue was embedded by scrapingeither ½ the cells on the plate or else all cells on the plate into 3.0mL of melted GN6 agarose medium (N6 medium, 2.0 mg/L 2,4-D, 30 g/Lsucrose, 100 mg/L myo-inositol, 7 g/L Sea Plaque agarose, pH 5.8,autoclaved for only 10 minutes at 121° C.) containing 1 mg/L bialaphos.The tissue was broken up and the 3 mL of agarose and tissue were evenlypoured onto the surface of a 100×15 mm plate of GN6 (1H) or GN6D (1H)medium. This was repeated for all remaining plates. Once embedded,plates were individually sealed with Nescofilm® or Parafilm M®, and thencultured for 1 week at 28° C. in dark boxes.

Putatively transformed isolates were typically first visible 5-8 weekspost-transformation. Any potential isolates were removed from theembedded plate and transferred to fresh selection medium of the sameconcentration in 60×20 mm plates. If sustained growth was evident afterapproximately 2 weeks, an event was deemed to be resistant and wassubmitted for molecular analysis.

Regeneration was initiated by transferring callus tissue to acytokinin-based induction medium, 28 (1H), containing 1 mg/L bialaphos,MS salts and vitamins, 30.0 g/L sucrose, 5 mg/L BAP, 0.25 mg/L 2,4-D,2.5 g/L Gelrite; pH 5.7. Cells were allowed to grow in low light (13μEm⁻²s⁻¹) for one week then higher light (40 μEm⁻²s⁻¹) for another weekbefore being transferred to regeneration medium, 36 (1H), which wasidentical to 28 (1H) except that it lacked plant growth regulators.Small (3-5 cm) plantlets were removed and placed into 150×25-mm culturetubes containing selection-free SHGA medium (Schenk and Hildebrandtbasal salts and vitamins, 1972; 1 g/L myo-inositol, 10 g/L sucrose, 2.0g/L Gelrite, pH 5.8). Once plantlets developed a sufficient root andshoot system, they were transplanted to soil in the greenhouse.

7.6—WHISKERS-Mediated Transformation Using Haloxyfop Selection.

DNA delivery parameters for direct selection on “fops” were identical tothe bialaphos selection procedure except that 85 μg of pDAB3403 and 85μg of construct containing a GFP (Green Fluorescent Protein) reportergene were co-transformed together, and only 3 mL of suspension wasfiltered onto GN6 medium following the 2-hour recovery.

After 0-7 days on GN6 selection-free medium, filter papers weretransferred to 60×20-mm plates of GN6 medium containing 2 mg/L 2,4-Dplus 50, 100, or 200 nM R-haloxyfop acid. Plates were placed in boxesand cultured for one additional week. After one week, the tissue wasembedded by scraping all cells from the plate into 3.0 mL of melted GN6agarose medium containing the same concentration of selection agent asin the previous transfer. All steps afterward were identical to the PATselection/regeneration protocol except that 100 nM R-haloxyfop acid wasincluded in the regeneration media instead of 1 mg/L bialaphos.

7.7—Results. Multiple experiments testing various levels of haloxyfopand cyhalofop were initiated, and 47 isolates were recovered from directselection. A subset of the callus events were submitted for screeningusing PCR and Western analyses. Following these expression data, 21 leadevents were submitted for Southern analysis. Results using NcoI, aunique cutter, to obtain integration data following probing with AAD-1(v3), unequivocally demonstrate stable integration of AAD-1 (v3)following Whiskers-mediated transformation coupled with “fop” selection.

7.8—Quantitative Demonstration of In Vitro Tolerance from AAD-1 (v3)Expressing Callus Events from Bialaphos Selection

Ninety-seven callus isolates recovered from bialaphos selection weresubmitted for PAT copy number via Invader analysis and AAD-1 (v3) PTUanalysis via PCR (see Example 7.10). AAD-1 (v3) protein expression usingWestern blot/Sandwich ELISA (Example 11) was completed on a subset ofthe events. A summary is described in Table 21 below. At least 15 Toplants were regenerated from each of these events and sent for spraytesting and seed production.

TABLE 21 PCR for AAD-1 (v3) Callus Event PAT Copy # PTU Western 3404-0012 + + 3404-006 2 + + 3404-013 3 + + 3404-017 1 + + 3404-020 3 + nd3404-022 2 + + 3404-025 2 + + 3404-027 3 + + 3404-031 1 + + 3404-033 2 +nd 3404-036 3 + + 3404-044 3 + + 3404-050 3 + + 3404-053 3 + + 3404-0742 + + 3404-082 2 + +

A smaller subset of these events was assessed in dose-response studiesin comparison to a non-transformed control. A range of concentrations ofhaloxyfop (from Gallant Super formulation), up to 400 nM, were tested.Dose response data for one event, 3404-006, was generated using thegeneral methods of Example 7.4, and is shown in FIG. 13. Thisdemonstrates that event 3404-006 showed no significant reduction incallus growth at haloxyfop concentrations up to 400 nM whereasnon-transgenic corn callus tissue growth was inhibited at this rate.These data have not been normalized to account for inherent growthdifferences that are not related to the expression of the transgene.

7.9—WHISKERS-Mediated Transformation Using Imazethapyr Selection.

The ZmUbi1/AAD-1 (v3)/ZmPer5 cassette was removed from pDAB3404 withAscI/SwaI and inserted into pDAB2212 to create the AAD-1 (v3) and AHASWhiskers transformation vector pDAB3415, which is also referred to aspDAS1283) Once completed, this construct was transformed into maize viasilicon carbide whiskers-mediated transformation as described in Example7.4 except 2 mL of cells were filtered, followed by a 7-day recovery onGN6 medium followed by selection on media containing 3 μM imazethapyrfrom Pursuit® DG herbicide. Following Invader analysis, 36 events wereidentified that contained the AAD-1 (v3) and the AHAS genes.

Fifty-three corn calli events from these transformations were tested inboth ELISA and Western Blotting experiments for their AAD-1 (v3)expression—a subset of the data is shown below. For the events listed inTable 22, the expression levels of those detected positive ranged from90 to over 1000 ppm of total soluble proteins.

TABLE 22 AAD-1 Event ID Expression Western Number Level (ppm) Blot1283[1]-001 2 − 1283[1]-002 206 +++ 1283[1]-003 1 − 1283[1]-004 90 +++1283[1]-005 1 − 1283[1]-006 105 +++ 1283[1]-007 212 ++ 1283[1]-008 114 ±1283[1]-009 305 + 1283[1]-010 2 − 1283[1]-011 4 − 1283[1]-012 200 +++1283[1]-013 134 +++ 1283[1]-014 4 − 1283[1]-015 194 +++ 1283[1]-016 4 −1283[1]-017 196 +++ 1283[1]-018 3 − 1283[1]-019 178 + 1283[1]-020 260 ++1283[1]-021 144 +++ 1283[1]-022 140 +++ 1283[1]-023 191 +++ 1283[1]-024392 ++ 1283[1]-025 368 ++ 1283[1]-026 14 − 1283[1]-027 1006 ++ NegControl 3 − Neg Control 3 − Standard (0.5 μg/mL) ++ Standard (5 μg/mL)++++

7.10—Molecular Analysis: Maize Materials and Methods.

7.10.1—Tissue harvesting DNA isolation and quantification. Fresh tissuewas placed into tubes and lyophilized at 4° C. for 2 days. After thetissue was fully dried, a tungsten bead (Valenite) was placed in thetube and the samples were subjected to 1 minute of dry grinding using aKelco bead mill. The standard DNeasy DNA isolation procedure was thenfollowed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA wasthen stained with Pico Green (Molecular Probes P7589) and read in thefluorometer (BioTek) with known standards to obtain the concentration inng/μ1.

7.10.2—Invader assay analysis. The DNA samples are diluted to 20 ng/μ1then denatured by incubation in a thermocycler at 95° C. for 10 minutes.Signal Probe mix was then prepared using the provided oligo mix andMgCl₂ (Third Wave Technologies). An aliquot of 7.5 μl was placed in eachwell of the Invader assay plate followed by an aliquot of 7.5 μl ofcontrols, standards, and 20 ng/μ1 diluted unknown samples. Each well wasoverlaid with 15 μl of mineral oil (Sigma). The plates are thenincubated at 63° C. for 1 hour and read on the fluorometer (Biotek).Calculation of % signal over background for the target probe divided bythe % signal over background internal control probe will calculate theratio. The ratio of known copy standards developed and validated withSouthern blot analysis was used to identify the estimated copy of theunknown events.

7.10.3—Polymerase chain reaction. A total of 100 ng of total DNA wasused as the template. 20 mM of each primer was used with the Takara ExTaq PCR Polymerase kit (Mirus TAKRROO1A). Primers for the AAD-1 (v3) PTUwere (Forward—ATAATGCCAGC CTGTTAAACGCC) (SEQ ID NO:25) and(Reverse—CTCAAGCATATGAATGACCT CGA) (SEQ ID NO:26). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 63° C. for 30 seconds, and 72° C. for 1 minute and 45seconds followed by 72° C. for 10 minutes. Primers for Coding Region PCRAAD-1 (v3) were (Forward—ATGGCTCATGCTGCCCTCAGCC) (SEQ ID NO:27) and(Reverse—CGGGC AGGCCTAACTCCACCAA) (SEQ ID NO:28). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute and 45seconds followed by 72° C. for 10 minutes. PCR products were analyzed byelectrophoresis on a 1% agarose gel stained with EtBr.

7.10.4—Southern blot analysis. Southern blot analysis was performed withtotal DNA obtained from Qiagen DNeasy kit. A total of 2 μg of DNA wassubjected to an overnight digestion of Afl II and also EcoRV forpDAB3404, NcoI for pDAB3403, and SpeI for pDAB1421 to obtain integrationdata. After the overnight digestion an aliquot of ˜100 ng was run on a1% gel to ensure complete digestion. After this assurance the sampleswere run on a large 0.85 agarose gel overnight at 40 volts. The gel wasthen denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. The gel wasthen neutralized in 0.5 M Tris HCl, 1.5 M NaCl pH of 7.5 for 30 minutes.A gel apparatus containing 20×SSC was then set up to obtain a gravitygel to nylon membrane (Millipore INYC00010) transfer overnight. Afterthe overnight transfer the membrane was then subjected to UV light via acrosslinker (Stratagene UV stratalinker 1800) at 1200 X100 microjoules.The membrane was then washed in 0.1% SDS, 0.1 SSC for 45 minutes. Afterthe 45 minute wash, the membrane was baked for 3 hours at 80° C. andthen stored at 4° C. until hybridization. The hybridization templatefragment was prepared using the above coding region PCR using plasmidpDAB3404. The product was run on a 1% agarose gel and excised and thengel extracted using the Qiagen (28706) gel extraction procedure. Themembrane was then subjected to a pre-hybridization at 60° C. step for 1hour in Perfect Hyb buffer (Sigma H7033). The Prime it RmT dCTP-labelingrxn (Stratagene 300392) procedure was used to develop the p32 basedprobe (Perkin Elmer). The probe was cleaned up using the Probe Quant.G50 columns (Amersham 27-5335-01). Two million counts CPM were used tohybridize the southern blots overnight. After the overnighthybridization the blots were then subjected to two 20 minute washes at65° C. in 0.1% SDS, 0.1 SSC. The blots were then exposed to filmovernight, incubating at −80° C.

Example 8—In Vivo Tolerance and Field Tolerance Data Generated fromPAT-Selected (pDAB3404) AAD-1 (v3) Events

8.1—Tolerance of T₀ Corn Plants to AOPP Herbicides.

If more than 15 clone plants per event were successfully regenerated,then extra plants were transferred to the greenhouse for preliminarytolerance screening with postemergence-applied AOPP herbicides on T₀corn plants. Greenhouse-acclimated plants were allowed to grow until 2-4new, normal looking leaves had emerged from the whorl (i.e., plants hadtransitioned from tissue culture to greenhouse growing conditions).Plants were grown at 27° C. under 16 hour light: 8 hour dark conditionsin the greenhouse. Plants were then treated with commercial formulationsof one of three AOPP herbicides: Assure® II (DuPont), Clincher* (DowAgroSciences), or Gallant Super* (Dow AgroSciences) for quizalofop,cyhalofop, or haloxyfop, respectively. Herbicide applications were madewith a track sprayer at a spray volume of 187 L/ha, 50-cm spray height,and all sprays contained 1% v/v Agridex crop oil concentrate adjuvant.The number of clones of each event varied from week to week due to therate of regeneration and acclimation of each event. Overall, an attemptwas made to treat representative clones of each event with a range ofherbicide doses ranging from 1× lethal dose (˜⅛× field dose) up to 8×field doses (64× lethal dose). A lethal dose is defined as the rate thatcauses >95% injury to the Hi-II inbred. Hi-II is the genetic backgroundof the transformants of the present invention.

AOPP's are generally very potent corn-killing herbicides. Three to fourleaf Hi-II corn grown from seed is effectively killed (>95% injury) with8.8, 62.5, and 4.4 g ae/ha of haloxyfop, cyhalofop, and quizalofop,respectively. Each AAD-1 (v3)-transformed line tested survived aminimally lethal dose of each AOPP herbicide tested. In fact, most linestested survived with no visible injury (14 DAT) even when treated withan 8× field dose (64× lethal dose) of quizalofop. Several individualclones from events “017” and “038,” however, did incur significantinjury at elevated rates. This could be a function of lower geneexpression due to how or where the gene was inserted.

The high level of AOPP tolerance was demonstrated in most events, evenwhen applications were made to plants just coming out of tissue culture(To stage). Significantly, this tolerance was shown for all three AOPPherbicides and likely will extend to all AOPP herbicides as previouslyshown for AAD-1 in vitro.

FIG. 14 shows the responses of several AAD-1 (v3)-transformed andnon-transformed event clones to lethal doses of two AOPP herbicides(haloxyfop and quizalofop) applied 1 week prior.

Table 23 shows data for the responses of selected AAD-1 (v3)-transformedT₀ corn events to three AOPP herbicides applied postemergence.

TABLE 23 Selected AAD-1 (v3)-transformed T0 corn events response tothree AOPP herbicides applied postemergence. Haloxyfog* Cyhalofop**Quizalofop*** % Injury Construct Event Clone g ae/ha g ae/ha g ae/ha 14DAT 3404 001 016 8.8 0 3404 001 018 62.5 0 3404 001 017 8.8 0 3404 001019 8.8 30 3404 001 020 35 0 3404 017 018 35 0 3404 017 019 35 0 3404017 020 70 0 3404 017 021 70 0 3404 017 022 140 0 3404 017 023 140 303404 017 024 280 30 3404 017 025 280 20 3404 022 019 8.8 0 3404 022 02017.5 0 3404 022 016 17.5 0 3404 022 024 62.5 0 3404 022 018 125 0 3404022 021 8.8 0 3404 022 017 17.5 0 3404 022 022 35 0 3404 022 023 70 03404 033 012 35 0 3404 033 013 70 0 3404 033 014 70 0 3404 033 015 140 03404 033 016 280 0 3404 038 016 8.8 0 3404 038 018 62.5 0 3404 038 0178.8 0 3404 038 019 35 70 3404 038 020 35 80 3404 038 021 70 80 3404 038022 70 80 (lethal dose = (lethal dose = (lethal dose = 8.8 g ae/ha) 62.5ae/ha) 4.4 g ae/ha) *Gallant super^(#) + 1% COC (v/v) **Clincher ^(#) +1% COC (v/v) ***Assure II + 1% COC (v/v) *Trademark of Dow AgroSciences,LLC

8.2—Field Tolerance of pDAB3404 T₁ corn Plants to Quizalofop 2,4-D, andGlufosinate Herbicides.

Two field trials were established at field stations in Hawaii andIndiana. Corn seed from inbred T₁ plants were utilized to evaluatesixteen AAD1 event lines for tolerance against quizalofop and 2,4-D.Three non-transformed hybrids were included for comparison purposes. Thehybrid Hi-II×5XH571 is of the same parentage as the AAD-1 (v3) eventlines. The hybrid Croplan 585SR is a sethoxydim resistant line.

The experimental design was a split-plot with four replications. Themain plot was herbicide treatment and the sub-plot was AAD-1 (v3) eventor comparison hybrid. Plots were one row by 3.7 meters withapproximately twenty-five seeds planted in each row. For AAD1 events,seeds from a different lineage within the event were planted in eachreplicate.

Glufosinate at 560 g ai/ha was applied to AAD-1 (v3) plots at the V2stage to eliminate non-transformed plants. Experimental treatmentsincluded commercial formulations of quizalofop applied at 70 and 140 gae/ha, 2,4-D (dimethylamine salt) at 560 and 1120 g ae/ha, and anuntreated control. Treatments were applied using backpack broadcast boomequipment delivering 187 L/ha carrier volume at 130-200 kpa pressure.Quizalofop treatments were applied at the V3-V4 corn stage and 2,4-Dtreatments were applied at the V5-V6 stage.

Quizalofop treated plots were visually assessed for crop injury at oneand three weeks after application (WAA) using a 0-100% scale, where 0equals no injury and 100 equals complete death. 2,4-D treated plots werevisually assessed for plant leaning at 2 days after application (DAA)using 0-100% scale where 0 equals no leaning from any plant and 100equals all plants prone. Additionally, 2,4-D plots were visuallyassessed at 3-4 WAA for brace root deformation using a 0-10 scale.

8.2.1—Results.

AAD-1 (v3) event response to the highest rates tested of quizalofop and2,4-D are shown in Table 24. These rates represent approximately twicethe normal commercial use rates. Non-transformed hybrids were severelyinjured (80-100%) by quizalofop at 70 g ae/ha including the sethoxydimresistant line, although it displayed slightly better tolerance than theother two hybrids. All AAD-1 (v3) events except one lineage of event3404.001 displayed excellent tolerance to quizalofop at 70 g ae/ha. Novisible symptoms were observed on the AAD-1 (v3) events except with theevents noted above.

TABLE 24 Treatment (rate)= 2,4-D 2,4-D Resistance amine amineSegregation Ratios Quizalofop (1120 g (1120 g following Liberty (140 gae/ha) ae/ha) ae/ha) Analysis Results Spray Evaluation= % AAD-1 % Injury3 Leaning Braceroot copy WAA 2 DAA Deformation number AAD-1 AAD-1 T₁ T₂(0-100 (0-100 3-4 WAA leaf Leaf Leaf Population Population Event orscale) scale) (0-10 scale) (Southern Western ELISA Average AverageHybrid IN HI IN HI IN HI analysis) Blot T₀ T₀ IN HI HI 3404.001 25 25 05 1 2 3 +++ +++ 40% 47% X 3404.006 0 0 0 0 0 0 1 ++ +++ 33% 26% X3404.013 0 0 0 0 0 0 1 + +++ 63% 58% X 3404.017 0 0 1 0 0 0 2 +/− ++ 48%47% X 3404.020 0 0 0 0 0 0 2 ++ +++ 50% 51% X 3404.022 0 0 1 0 0 0 1 +++ 51% 57% 76%* 3404.025 0 0 0 0 0 0 2 +++ ++++ 55% 59% X 3404.027 0 0 30 0 0 5 + ++ 51% 50% X 3404.031 0 0 1 0 0 0 1 ++ ++++ 47% 43% 61%*3404.033 0 0 0 0 0 0 2 or 3 + ++ 52% 49% X 3404.036 0 0 0 0 0 0 4 ++ +++52% 48% X 3404.044 0 0 1 0 0 1 1 + +/− 50% 48% X 3404.050 0 0 0 1 0 1 2nd nd 38% 28% X 3404.053 0 0 1 0 0 0 2 +++ +++ 48% 56% X 3404.074 0 0 00 0 0 1 ++ +++ 53% 52% 73%* 3404.082 0 0 0 0 0 1 3 nd nd 38% 36% X DK493100 100 20 23 8 9 HI-II X 5XH571 100 100 13 34 7 9 CROPLAN 585SR 80 9611 33 9 9 *Fits single locus dominant trait segregation as determined bychi square analysis (P > 0.05)

2,4-D at the 1120 g ae/ha rate caused significant levels (11-33%) ofepinastic leaning in the non-transformed hybrids, a normal response whenapplied beyond the V4 growth stage. Little or no leaning was observedwith all AAD-1 (v3) events except one lineage of 3404.001 (Indianalocation only) where moderate levels (5-13%) of leaning occurred.

Brace roots of non-transformed hybrids were severely deformed (rating of9 on a 0-10 scale) by 2,4-D at the 1120 g ae/ha rate. Again, this is anormal response to 2,4-D applied beyond the V4 growth stage. As with theleaning response, little or no brace root injury was observed with allAAD-1 (v3) events except one lineage of 3404.001.

Similar trends occurred with lower tested rates of quizalofop and 2,4-Dalthough at reduced but still significant response levels in thenon-transformed hybrids (data not shown).

These results indicate that most AAD-1 (v3) transformed event linesdisplayed a high level of resistance to quizalofop and 2,4-D at ratesthat were lethal or caused severe epinastic malformations tonon-transformed corn hybrids. See also FIG. 16.

8.2.2—Expected Mendelian segregation ratios on three T₂ events. Plantsfrom individual lineages of each event were randomly self-pollinated inthe field. T₂ seed were hand harvested at physiological maturity. Basedon single gene copy number (see Table 24 above) and overall performancein T₁ generation (segregation, herbicide tolerance, and vigor), threeevents (022, 031, and 074) were chosen for further evaluation in thefield. Breeding rows of each event were planted using a precision coneplanter each consisting of 2500-3000 seeds. At the V2 growth stage, allAAD-1 (v3) lines were sprayed with 140 g ae/ha quizalofop (Assure® II)using a backpack sprayer as previously described. This rate rapidlykilled all “null” (untransformed) segregants. Each event had asegregation ratio consistent with Mendelian inheritance of a singlelocus, dominant gene (3 resistant:1 sensitive, or 75% survival) (seeTable 24). Homozygotes and hemizygotes from event 74 were identified byzygosity testing (refer to AAD-1 (v3) Invader assay description forcorn). Hemizygous plants were removed and homozygous AAD-1 (v3) plantswere crossed with BE1146 corn inbred introgressed and homozygous forglyphosate resistance trait, NK603, creating a homogeneous F₁ hybridseed that is hemizygous for glyphosate resistance, AAD-1 (v3), andglufosinate resistance.

8.3—Stacking of AAD-1 (v3) and PAT with Glyphosate Resistance Genes inCorn.

Homozygous T₂ AAD-1 (v3)/PAT corn plants were crossed with glyphosateresistant corn plants producing F₁ seed containing AAD-1 (v3), PAT, andglyphosate resistance genes as described in the previous example.

F₁ seeds were planted individually into 3-inch pots prepared withMetro-Mix® 360 growing medium (Sun Gro Horticulture). The pots wereinitially subirrigated with Hoagland's solution until wet, then allowedto gravity drain, and grown at 27° C. under 16 hour light:8 hour darkconditions in the greenhouse. For the remainder of the study the plantswere subirrigated with deionized water.

Plants were allowed to grow until 2-4 leaves had emerged from the whorl.At this point herbicide applications were made with a track sprayer at aspray volume of 187 L/ha, 50-cm spray height. The plants were sprayedwith rates of 2,4-D DMA, glyphosate, glufosinate, and variouscombinations of the three. All applications were formulated in 200 mMHepes buffer (pH 7.5). In spray applications where glufosinate waspresent the treatment was formulated with the addition of 2% w/vammonium sulfate.

At 3 and 14 days after treatment (DAT) plants were evaluated. Plantswere assigned injury rating with respect to stunting, chlorosis, andnecrosis. Plants assigned an injury rating of 90% or above areconsidered dead. Results of the study at 14 DAT can be seen in Table 25.

TABLE 25 % Injury at 14 DAT Hi II X Field 5XH751 RR/PAT/AAD1 Rate AveAve Untreated control — 0 0 840 g ae/ha glyphosate 1X 98 0 1680 g ae/haglyphosate 2X 100 0 3360 g ae/ha glyphosate 4X 100 0 560 g ae/ha 2,4-DDMA 1X 10 0 1120 g ae/ha 2,4-D DMA 2X 14 0 2240 g ae/ha 2,4-D DMA 4X 290 470 g ae/ha glufosinate 1X 80 0 940 g ae/ha glufosinate 2X 90 3 1880 gae/ha glufosinate 4X 96 15 840 g ae/ha glyphosate + 560 g ae/ha 2,4-DDMA 1X + 1X 96 1 1680 g ae/ha glyphosate + 1120 g ae/ha 2,4-D DMA 2X +2X 100 2 3360 g ae/ha glyphosate + 2240 g ae/ha 2,4-D DMA 4X + 4X 100 1470 g ae/ha glufosinate + 560 g ae/ha 2,4-D DMA 1X + 1X 89 5 940 g ae/haglufosinate + 1120 g ae/ha 2,4-D DMA 2X + 2X 91 10 1880 g ae/haglufosinate + 2240 g ae/ha 2,4-D DMA 4X + 4X 97 13 840 g ae/haglyphosate + 470 g ae/ha glufosinate 1X + 1X 90 5 1680 g ae/haglyphosate + 940 g ae/ha glufosinate 2X + 2X 98 15 3360 g ae/haglyphosate + 1880 g ae/ha glufosinate 4X + 4X 100 15

This study demonstrated that the AAD-1 (v3) gene in corn can be stackedwith a glyphosate resistance gene and a glufosinate resistance gene toprovide robust field-level tolerance to 2,4-D, glyphosate, andglufosinate alone or in tank mix combinations.

8.3.1—Resistance of AAD-1 (v3) corn using a tank mix of 2,4-D DMA andquizalofop. T₂BC₁ seeds of hemizygous event number 3404-025.001R/R001Bulked.001.S058 were planted individually into 3-inch pots prepared withMetro-Mix® 360 growing medium. The pots were initially sub-irrigatedwith Hoagland's solution until wet, then allowed to gravity drain, andgrown at 27° C. under 16 hour light:8 hour dark conditions in thegreenhouse. For the remainder of the study the plants were sub-irrigatedwith de-ionized water.

Plants were allowed to grow until V1 stage in the greenhouse. At thispoint the plants were selected with 560 g ae/ha Assure® II with theaddition of 1% Agridex crop oil concentrate in 200 mM Hepes buffer withthe research track sprayer set at 187 L/ha. Plants were allowed 4 daysto show symptoms of the selection. All plants were uninjured. Herbicideapplications were made with a track sprayer at a spray volume of 187L/ha, 18-in spray height. All applications were formulated in 200 mMHepes buffer (pH 7.5) with the addition of 1% v/v Agridex.

At 3 and 14 days after treatment (DAT) plants were evaluated. Plantswere assigned injury rating with respect to stunting, chlorosis, andnecrosis. Plants assigned an injury rating of 90% or above areconsidered dead. Plants from this particular lineage had 0% injury at 14DAT for all tank mixed combinations, while the wild-type had 100%injury. These results indicate AAD-1 (v3) not only provides robust fieldlevel resistance to 2,4-D and quizalofop individually, but also toexaggerated rates of multiple combinations of the two chemistries. Onecould logically expect to implement novel weed control measures withcombinations of phenoxy auxins and AOPP graminicides in corn (or othercrops transformed with AAD-1) not previously enabled by a single geneHTC.

8.3.2—Tolerance of (pDAB3403) To corn plants to quizalofop herbicide. Atarget of approximately eight T₀ plant clones from each of 17 eventswere regenerated and transferred to the greenhouse for preliminarytolerance screening with postemergence-applied discriminating rate ofquizalofop herbicide applied by track sprayer at 35 g ae/ha (1× fieldrate, 4× lethal dose) to 3-leaf, greenhouse-adapted T₀ corn plants usingtrack sprayer conditions previously described. Plants were rated asResistant or Sensitive 7 days after treatment. Control, non-transgeniccorn was included with each spray application. Two events, Event 014 and047, had two or more T₀ clones sensitive to 35 g ae/ha quizalofop,indicating an unexpected level of sensitivity for this event. The 15other events showed stable integration, protein expression, and theability to tolerate a 4× lethal dose of quizalofop at the whole plantlevel.

8.3.3 Expression of AAD-1 (v3) with Respect to Quizalofop Tolerance.

Three different T₂ lineages from 3404 transformations that werepre-screened with Liberty® (as described previously) to remove nullswere chosen to compare their tolerance to quizalofop with respect totheir AAD-1 (v3) expression. Expression was measured at 14 DAT (data notshown) and at 30 DAT (see FIG. 15.). The highest tolerance line, event3404-074, always expressed with a higher amount of AAD-1 (v3) than theother two events at 1× and higher field rates. This data concludes thatcorn expressing AAD-1 (v3) can be protected from quizalofop injury atthe highest level tested (2,240 g/ha), which is 16 times the 1× fielddose of 35 g/ha. In addition, the expression level was consistentthroughout the period of the experiment.

Example 9—Agrobacterium-Mediated Transformation of Maize with AAD-1 (v3)

9.1—Plant Material.

Seeds of a “High II” (i.e., Parent A and B) F₁ cross (Armstrong et al.,1991) are planted directly into 5 gallon-pots containing 95:5 Metro-Mix®360: Mineral soil. The plants are grown in the greenhouse with a 16 hourphotoperiod supplemented by a combination of high pressure sodium andmetal halide lamps.

9.2—Tissue Source.

For obtaining immature Hi-II (F2) embryos, controlled sib-pollinationswere performed. On the day of pollination, actively shedding tassels arebagged, and fresh pollen is collected and applied carefully onto thesilks. Immature embryos were isolated as described in Example 7.2.

9.3—Preparation of a Superbinary Vector.

Construction of an Agrobacterium construct, pDAB2272, containing theAAD-1 (v3) gene in combination with the AHAS selectable marker gene wasaccomplished by isolating the 3443 base pair NotI fragment from pDAB3404containing ZmUbi1 v2/AAD-1 (v3)/ZmPer5 v2 and inserting it into the NotIsite of pDAB8549. The resulting plasmid contains the ZmUbi1 v2/AAD-1(v3)/ZmPer5 v2 and the OsAct1 v2/AHAS v3/ZmLip v1 cassettes flanked bynon-identical MAR regions in the direct orientation. This wassubsequently transformed into LBA4404/pSB1 to create the superbinaryvector, which was named pDAB3602 but was also referred to as pDAS1421.

9.4—Bacterial Supply.

All transformations use the “Super Binary” vector from Japan Tobaccodescribed in U.S. Pat. No. 5,591,616 (“Method for TransformingMonocotyledons”). To prepare the Agrobacterium suspension for treatment,1-2 loops of pDAS1421 recombinant bacteria from a YP streak plate wasput into 5 ml of LS-inf. Mod medium (LS Basal Medium (Linsmaier andSkoog, 1965), N6 vitamins, 1.5 mg/L 2,4-D, 68.5 g/L sucrose, 36.0 g/Lglucose, 6 mM L-proline, pH 5.2). The mixture was vortexed until auniform suspension was achieved. The bacterial concentration was takenusing a Klett-Summerson Photoelectric Colorimeter by reading the densityof the solution. The solution was adjusted to a concentration of Klett200 (˜1×10⁹ cfu/ml) and 100 μM actetosyringone added to the solution.

9.5—Infection and Cocultivation.

The immature embryos are isolated directly into a microfuge tubecontaining 2 ml LS-inf. Mod liquid medium. Each tube, containing ˜100embryos, is vortexed for 3-5 sec. The medium is removed and replacedwith fresh liquid medium and the vortex is repeated. The liquid mediumis again removed and this time replaced with an Agrobacterium solutionat the Klett 200 concentration. The Agrobacterium and embryo mixture isvortexed for 30 sec. Following a 5 minute incubation at roomtemperature, the embryos were transferred to LS-As Mod medium (LS BasalMedium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 g/L sucrose, 6 mM L-proline,0.85 mg/L AgNO₃,1, 100 μM actetosyringone, 3.0 g/L Gelrite, pH 5.8) fora 5-day co-cultivation at 25° C.

9.6—Dose Response Using Immature Embryos.

Dose response studies were initiated using immature embryos treated withAgrobacterium strain LBA4404 lacking a plasmid as described previously.Once treated, embryos were allowed to co-cultivate for 5 days at 25° C.and were then transferred to selection media containing various levelsof R-haloxyfop or R-cyhalofop. Embryos were also transferred to mediacontaining 1 mg/L bialaphos and 100 nM imazethapyr as negative controls.Embryos were scored for % embryogenic callus formation after 2 weeks,and then again after 4 weeks. Embryos were tested on R-haloxyfop levelsup to 30 nM; however, insufficient reduction of callus formation wasseen at the highest levels, so higher concentrations (50-100 nM) wereused during transformation experiments. Data from embryos grown oncyhalofop-containing media is shown in FIG. 17.

9.7—Selection.

After co-cultivation, the embryos were moved through a 2-step selectionscheme after which transformed isolates were obtained. For selection,LSD Mod medium (LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 0.5 g/LMES, 30.0 g/L sucrose, 6 mM L-proline, 1.0 mg/L AgNO₃, 250 mg/Lcephotaxime, 2.5 g/L Gelrite, pH 5.7) was used along with one of twoselection levels of either haloxyfop, cyhalofop, or imazethapyr.Throughout the selection phase, the embryos are cultured in the dark at28° C. The embryos were first transferred to an initial level ofselection (50-100 nM R-haloxyfop or 300 nM R-cyhalofop) for 14 days,then moved up to a higher selection level (250-500 nM R-haloxyfop acidor 1.5 μM cyhalofop) at a rate of 5 embryos/plate. A subset of embryoswere similarly stepped-up from 100 to 500 nM imazethapyr from Pursuit®DG as a positive control. Pursuit® is used as the chemical selectionagent when the AHAS gene is used, based on U.S. Pat. No. 5,731,180.Tissue was transferred at biweekly intervals on the same medium untilembryogenic colonies were obtained. These colonies were maintained onthe high selection pressure for the remainder of the culture period. Therecovered transgenic colonies were bulked up by transferring to freshselection medium at 2-week intervals for regeneration and furtheranalysis.

9.8.—Regeneration and Seed Production.

For regeneration, the cultures are transferred to 28 “induction” mediumand 36 “regeneration” medium as described previously containing either100 nM R-haloxyfop or 1.5 μM cyhalofop for differentiation of plantlets.When plantlets were established, they were transferred to SHGA tubes toallow for further growth and development of the shoot and roots asdescribed previously. Controlled pollinations for seed production wereconducted as described previously.

9.9—Event Recovery and Analysis Particulars; Whole Plant Screening of toCorn Lineages Containing AAD-1 (v3) and AHAS (pDAS1421).

Seventy-two Agrobacterium-transformed events were selected on variouslevels of R-haloxyfop acid and R-cyhalofop acid in vitro. Twenty-twocallus samples were analyzed by Southern blot analysis for stableintegration of AAD-1 (v3) into the genome as described previously. Tensingle copy events, as indicated in Table 26, were chosen to beregenerated.

TABLE 26 Southern Assessment of T₀ lineages In vitro Copy # Western Blotresistant to 35 g ae/ha Selection agent (callus) (AAD-1) T₀ quizalofopCorn event (nM) AAD-1 Callus Resistant Sensitive 1421[21]-016 50Haloxyfop 1 + 8 0 1421[22]-020 100 Haloxyfop 1 ++ 8 0 1421[22]-022 100Haloxyfop 1 + 8 0 1421[22]-023 100 Haloxyfop 1 ++ 8 0 1421[3]-036 100Haloxyfop 1 ++ 8 0 1421[4]-031 300 Cyhalofop 1 ++ 9 0 1421[4]-032 300Cyhalofop 1 ++ 8 0 1421[4]-033 300 Cyhalofop 1 ++ 12 0 1421[4]-034 300Cyhalofop 1 ++ 8 0 1421[4]-035 300 Cyhalofop 1 ++ 6 0 Events with morethan 1 copy were not taken to the greenhouse

A minimum six regenerated clonal lineages per event were moved to soilin the greenhouse and screened using a track sprayer as previouslydescribed to apply 35 g ae/ha quizalofop when 2-4 new, normal leaves hademerged (see section 8.3.3). Presence of AAD-1 protein on a Western blotcorrelated perfectly with herbicide resistance in the T₀ generationregardless of which AOPP herbicide was used for selection. There is nonegative impact of the second HTC gene (AHAS) on the function of theAAD-1 (v3).

Example 10—Purification of AAD-1 (v1) for Antibody Creation andBiochemical Characterization

All operations during purification were carried out at 4° C. Frozen orfresh E. coli cells from approximately 1 L culture, grown and induced asin Example 3, were re-suspended in 200 ml of extraction buffercontaining 20 mM Tris-HCl, 1 mM EDTA, and 2 ml of Protease InhibitorCocktail (Sigma), and disrupted by ultrasonication treatment on iceusing a Branson sonifier. The soluble extract was obtained bycentrifugation in a GSA rotor (Sorvall) at 12,000 rpm (24,000 g) for 20minutes. The supernatant was then loaded onto a Mono Q ion exchangecolumn (Pharmacia HR 10/10) equilibrated with 20 mM Tris-HCl, 1 mM EDTA,pH 8.0, and the column was washed with same buffer for 10 CV (80 ml).The protein was eluted with 80 ml of a 0 to 0.25 M NaCl linear gradientin column buffer, while 2 ml fractions were collected. The fractionscontaining AAD-1 (v1) were pooled and concentrated using MWCO 30 kDamembrane centrifugation spin columns (Millipore). The sample was thenfurther separated on a Superdex 200 size exclusion column (Pharmacia, XK16/60) with buffer containing 20 mM Tris-HCl, 0.15 M NaCl, and 1 mM DTT,pH 8.0 at a flow rate of 1 ml/min. Purification procedures were analyzedby SDS-PAGE, and protein concentration was determined by Bradford assayusing bovine serum albumin as standard.

Example 11—Recombinant AAD1 Purification and Antibody Production

Plasmid pDAB3203 containing the AAD-1 (v1) gene was maintained frozen at−80° C. in TOP10F′ cells (Invitrogen) as Dow Recombinant strain DR1878.For expression, plasmid DNA purified from TOP10F′ cell culture usingPromega's Wizard Kit (Fisher cat. #PR-A1460) was transformed into BL-21Star (DE3) cells (Invitrogen cat. #C6010-03) following manufacturer'sprotocol. After transformation, 50 μL of the cells were plated onto LBS/S agar plates, and incubated overnight at 37° C. All colonies from theentire agar plate were scraped into 100 mL LB in a 500 mL tribaffledflask and incubated at 37° C. with 200 rpm shaking for 1 hr. Geneexpression was then induced with 1 mM IPTG, and incubated for 4 hrs at30° C. with 200 rpm shaking. All 100 mL of culture was centrifuged at4000 rpm for 20 min. The supernatant were then discarded, and thepellets were resuspended in 200 mL of extraction containing 20 mMTris-HCl (pH 8.0), 1 mM EDTA, and 2 mL of Protease Inhibitor Cocktail(Sigma), and disrupted by ultrasonication treatment on ice using aBranson sonifier. The lysate was centrifuged at 24,000×g for 20 mM toremove cell debris. The supernatant containing the AAD-1 protein wasthen subjected to purification protocol.

All AAD-1 (v1) purifications were conducted at 4° C. as discussed inExample 10, unless otherwise stated. The cell lysate was loaded onto aMono Q ion exchange column (Pharmacia Cat. #HR 10/10) equilibrated with20 mM Tris-HCl (pH 8.0) 1 mM EDTA, followed by 80 mL of washing with thesame buffer. The proteins were eluted with 80 mL of a 0 to 0.25 M NaCllinear gradient in column buffer, while 2 mL fractions were collected.The fractions containing AAD-1 were pooled and concentrated using MWCO30 kDa membrane centrifugation spin columns (Millipore). The sample wasthen further separated on a Superdex 200 size exclusion column(Pharmacia, XK 16/60) with buffer containing 20 mM Tris-HCl (pH 8.0),0.15 M NaCl and 1 mM DTT. Protein concentration was determined byBradford assay using bovine serum albumin as standard.

Five milligrams purified AAD-1 (v1) was delivered to Zymed Laboratories,Inc. (South San Francisco, Calif.) for rabbit polyclonal antibodyproduction. The rabbit received 5 injections in the period of 5 weekswith each injection containing 0.5 mg of the purified protein suspendedin 1 mL of Incomplete Freund's Adjuvant. Sera were tested in both ELISAand Western blotting experiments to confirm specificity and affinitybefore affinity purification and horseradish peroxidase (HRP)conjugation (Zymed Lab Inc).

11.1—Extracting AAD-1 (v3) from Plant Leaves.

Approximately 50 to 100 mg of leaf tissue was cut into small pieces andput into microfuge tubes containing 2 stainless steel beads (4.5 mm;Daisy Co., cat. #145462-000) and 300 μL plant extraction buffer (PBScontaining 0.1% Triton X-100 and 10 mM DTT). The tubes were shaken for 4mM with a bead beater at maximum speed followed by centrifugation for 10mM at 5,000×g. The supernatant containing the plant soluble proteinswere analyzed for both total soluble protein (TSP) and AAD-1 (v3)concentrations.

11.2—Bradford Assay.

Total soluble protein concentration from plant leaf tissues weredetermined by Bradford assay using bovine serum albumin (BSA) asstandard. Five micro-liter of serially diluted BSA in PBS or plantextract was transferred to 96-well microtiter plate in triplicates. Forstandards, concentrations were ranged from 2000 to 15.6 μg/mL. Theprotein assay concentrate was first diluted 5 fold in PBS and 250 μL wasadded to each well and incubated at room temp for 5 mM Each opticaldensity (OD) was measured at 595 nm using a microplate reader. Theprotein concentration of each sample was extrapolated from standardcurve using the Softmax® Pro (ver. 4.0) (Molecular Devices).

11.3—Enzyme Linked Immuno-Sorbent Assay (ELISA).

The assay was conducted at room temperature unless otherwise stated. Onehundred micro-liter of purified anti-AAD-1 antibody (0.5 μg/mL) wascoated on 96-well microtiter well and incubated at 4° C. for 16 hours.The plate was washed four times with washing buffer (100 mM phosphatebuffered saline (PBS; pH 7.4) containing 0.05% Tween 20) using a platewasher, followed by blocking with 4% skim milk dissolved in PBS for 1hour. After washing, 100 μL standard AAD-1 of known concentrations orplant extract (see previous section) was incubated in the wells. Forstandard curve, purified AAD-1 concentrations ranged from 100 to 1.6ng/mL in triplicates. Plant extracts were diluted 5, 10, 20, and 40 foldin PBS and analyzed in duplicates. After 1 hour incubation, the platewas washed as above. One hundred micro-liter anti-AAD-1 antibody-HRPconjugate (0.25 ug/mL) was incubated in each well for 1 hour beforewashing. One hundred micro-liter HRP substrate, 1-Step™ Ultra TMB-ELISA(Pierce), was incubated in each well for 10 minutes before the reactionwas stopped by adding 100 μL 0.4N H2504. The OD of each well wasmeasured using a microplate reader at 450 nm. To determine theconcentrations of AAD-1 in plant extract, the OD value of duplicateswere averaged and extrapolated from the standard curve using theSoftmax® Pro ver. 4.0 (Molecular Devices).

For comparison, each sample was normalized with its TSP concentrationand percent expression to TSP was calculated.

11.4—Western Blotting Analysis.

Plant extracts or AAD-1 standards (5 and 0.5 μg/mL) were incubated withLaemmli sample buffer at 95° C. for 10 minutes and electrophoreticallyseparated in 8-16% Tris-Glycine Precast gel. Proteins were thenelectro-transferred onto nitrocellulose membrane using standardprotocol. After blocking in 4% skim milk in PBS, AAD-1 protein wasdetected by anti-AAD1 antiserum followed by goat anti-rabbit/HRPconjugates. The detected protein was visualized by chemiluminescencesubstrate ECL Western Analysis Reagent (Amersham cat. #RPN 21058).

Example 12—Tobacco Transformation

Tobacco transformation with Agrobacterium tumefaciens was carried out bya method similar, but not identical, to published methods (Horsch etal., 1988). To provide source tissue for the transformation, tobaccoseed (Nicotiana tabacum cv. Kentucky 160) was surface sterilized andplanted on the surface of TOB-medium, which is a hormone-free Murashigeand Skoog medium (Murashige and Skoog, 1962) solidified with agar.Plants were grown for 6-8 weeks in a lighted incubator room at 28-30° C.and leaves collected sterilely for use in the transformation protocol.Pieces of approximately one square centimeter were sterilely cut fromthese leaves, excluding the midrib. Cultures of the Agrobacteriumstrains (EHA101S containing pDAB721, AAD-1 (v3)+PAT), grown overnight ina flask on a shaker set at 250 rpm at 28° C., were pelleted in acentrifuge and resuspended in sterile Murashige & Skoog salts, andadjusted to a final optical density of 0.5 at 600 nm. Leaf pieces weredipped in this bacterial suspension for approximately 30 seconds, thenblotted dry on sterile paper towels and placed right side up on TOB+medium (Murashige and Skoog medium containing 1 mg/L indole acetic acidand 2.5 mg/L benzyladenine) and incubated in the dark at 28° C. Two dayslater the leaf pieces were moved to TOB+ medium containing 250 mg/Lcefotaxime (Agri-Bio, North Miami, Fla.) and 5 mg/L glufosinate ammonium(active ingredient in Basta, Bayer Crop Sciences) and incubated at28-30° C. in the light. Leaf pieces were moved to fresh TOB+ medium withcefotaxime and Basta twice per week for the first two weeks and once perweek thereafter. Four to six weeks after the leaf pieces were treatedwith the bacteria, small plants arising from transformed foci wereremoved from this tissue preparation and planted into mediumTOB-containing 250 mg/L cefotaxime and 10 mg/L Basta in Phytatray™ IIvessels (Sigma). These plantlets were grown in a lighted incubator room.After 3 weeks, stem cuttings were taken and re-rooted in the same media.Plants were ready to send out to the greenhouse after 2-3 additionalweeks.

Plants were moved into the greenhouse by washing the agar from theroots, transplanting into soil in 13.75 cm square pots, placing the potinto a Ziploc® bag (SC Johnson & Son, Inc.), placing tap water into thebottom of the bag, and placing in indirect light in a 30° C. greenhousefor one week. After 3-7 days, the bag was opened; the plants werefertilized and allowed to grow in the open bag until the plants weregreenhouse-acclimated, at which time the bag was removed. Plants weregrown under ordinary warm greenhouse conditions (30° C., 16 hour day, 8hour night, minimum natural+supplemental light=500 μE/m²s¹).

Prior to propagation, T₀ plants were sampled for DNA analysis todetermine the insert copy number. The PAT gene which was molecularlylinked to AAD-1 (v3) was assayed for convenience. Fresh tissue wasplaced into tubes and lyophilized at 4° C. for 2 days. After the tissuewas fully dried, a tungsten bead (Valenite) was placed in the tube andthe samples were subjected to 1 minute of dry grinding using a Kelcobead mill. The standard DNeasy DNA isolation procedure was then followed(Qiagen, DNeasy 69109). An aliquot of the extracted DNA was then stainedwith Pico Green (Molecular Probes P7589) and read in the florometer(BioTek) with known standards to obtain the concentration in ng/μl.

The DNA samples were diluted to 9 ng/μ1, then denatured by incubation ina thermocycler at 95° C. for 10 minutes. Signal Probe mix was thenprepared using the provided oligo mix and MgCl2 (Third WaveTechnologies). An aliquot of 7.5 μl was placed in each well of theInvader assay plate followed by an aliquot of 7.5 μl of controls,standards, and 20 ng/μ1 diluted unknown samples. Each well was overlaidwith 15 μl of mineral oil (Sigma). The plates were then incubated at 63°C. for 1.5 hours and read on the florometer (Biotek). Calculation of %signal over background for the target probe divided by the % signal overbackground internal control probe will calculate the ratio. The ratio ofknown copy standards developed and validated with southern blot analysiswas used to identify the estimated copy of the unknown events (Table27).

TABLE 27 Tobacco T0 events transformed with pDAB721 (AAD-1(v3) + PAT).PAT copy Coding ELISA (μg Relative number Region PCR AAD-1/ml toleranceT0 Event (Southern) for AAD-1 plant extract) spray with 2,4-D * 721(1)11 + 0.9 Medium 721(2)1 nd nd 0.6 Medium 721(2)2 5 + 0.3 Low 721(2)3 3 +2.6 Medium 721(2)5 5 + 4.1 Variable 721(2)6 3 + 0.5 Variable 721(2)8 5 +0.3 High 721(2)11 3 + n/a High 721(2)12 3 + 4.1 Medium 721(2)13 2 + 0.5Medium 721(2)14 5 + 0.2 High 721(2)16 4 + 3.2 Medium 721(2)17 3 + ndHigh 721(2)18 5 + nd High 721(2)19 >10 + nd Low 721(2)20 5 + nd Medium721(2)21 4 + nd High 721(2)22 7 + nd Medium 721(2)23 >10 + nd Variable721(3)003 3 + nd Variable 721(3)008 2 + nd High 721(3)012 1 + nd High721(3)4 2 + 0.5 High 721(3)5 9 + 3.3 High 721(3)6 4 + 7.1 Variable721(3)9 2 + 1   Low 721(3)10 3 + 0.6 High 721(3)11 7 + 6   Low 721(3)134 + 0.1 High 721(3)014 2 + 0.1 Medium nd = not done Legend: Relativetolerance * Injury at 3200 g ae/ha 2,4-D (14 DAT) Low >50% injury Medium20-50% injury High <20% injury Variable inconsistent

Copy number estimations were confirmed by Southern Analysis on severalevents. Southern blot analysis was performed with total DNA obtainedfrom Qiagen DNeasy kit. A total of 2 μgs of DNA was subjected to anovernight digest of NsiI and also HindIII for pDAB721 to obtainintegration data. After the overnight digestion an aliquot of ˜100 ngswas run on a 1% gel to ensure complete digestion. After this assurancethe samples were processed using same protocol as in Example 6 section11.

All events were also assayed for the presence of the AAD-1 (v3) gene byPCR using the same extracted DNA samples. A total of 100 ng of total DNAwas used as template. 20 mM of each primer was used with the Takara ExTaq PCR Polymerase kit (Minis TAKRROO1A). Primers for the Coding RegionPCR AAD-1 were (RdpAcodF ATGGCTCA TGCTGCCCTCAGCC) (SEQ ID NO:27) and(RdpAcodR CGGGCAGGCCTAACTCCACC AA) (SEQ ID NO:28). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 64° C. for 30 seconds, and 72° C. for 1 minute and 45seconds followed by 72° C. for 10 minutes. PCR products were analyzed byelectrophoresis on a 1% agarose gel stained with EtBr. Four to 12 clonallineages from each of 30 PCR positive events were regenerated and movedto the greenhouse.

A representative plant from each of 19 events was assayed for AAD-1 (v3)expression by ELISA methods previously described. All events assayedshowed detectable levels of AAD-1 (v3) (Table 27). Protein expressionvaried across events.

T₀ plants from each of the 30 events were challenged with a wide rangeof 2,4-D sprayed on plants that were 3-4 inches tall. Spray applicationswere made as previously described using a track sprayer at a sprayvolume of 187 L/ha. 2,4-D dimethylamine salt (Riverside Corp) wasapplied at 0, 50, 200, 800, or 3200 g ae/ha to representative clonesfrom each event mixed in deionized water. Each treatment was replicated1-3 times. Injury ratings were recorded 3 and 14 DAT. Every event testedwas more tolerant to 2,4-D than the untransformed control line KY160. Inseveral events, some initial auxinic herbicide-related epinasty occurredat doses of 800 g ae/ha or less. Some events were uninjured at this rate(equivalent to 1.5× field rate). All events suffered some leveltemporary auxinic damage 3 DAT when treated with 3200 g ae/ha. Some leafburning also occurred at this high rate due to the acidity of the spraysolution. Future trials at high 2,4-D rates were buffered. Response ofT₀ plants treated with 3200 g ae/ha 2,4-D (˜6× field rate) was used todiscern relative tolerance of each event into “low” (>50% injury 14DAT), “medium” (20-50% injury), “high” (<20% injury). Some events wereinconsistent in response among replicates and were deemed “variable”(Table 27).

Verification of High 2,4-D Tolerance.

Two to four T₀ individuals surviving high rates of 2,4-D were saved fromeach event and allowed to self fertilize in the greenhouse to give riseto T₁ seed. Two AAD-1 (v3) tobacco lines (event 721(2)-013.010 and721(3)-008.005) were chosen from the T₀ generation. The T₁ seed wasstratified, and sown into selection trays much like that of Arabidopsis(Example 6.4), followed by selective removal of untransformed nulls inthis segregating population with 280 g ai/ha glufosinate (PATselection). Survivors were transferred to individual 3-inch pots in thegreenhouse. These lines provided medium and high levels of robustness to2,4-D in the T₀ generation. Improved consistency of response isanticipated in T₁ plants not having come directly from tissue culture.These plants were compared against wildtype KY 160 tobacco. All plantswere sprayed with the use of a track sprayer set at 187 L/ha. The plantswere sprayed from a range of 70-4480 g ae/ha 2,4-D dimethylamine salt(DMA), R-Dichlorprop, and a 50/50 mix of the two herbicides. Allapplications were formulated in 200 mM Hepes buffer (pH 7.5). Eachtreatment was replicated 4 times. Plants were evaluated at 3 and 14 daysafter treatment. Plants were assigned injury rating with respect tostunting, chlorosis, and necrosis. The T₁ generation is segregating, sosome variable response is expected due to difference in zygosity. (Table28). No injury was observed at rates below 1× field rates (560 g ae/ha)for 2,4-D or R-dichlorprop in either event. Very little injury wasobserved even up to 8 times field rates (4480 g ae/ha) and this wasexhibited as stunting, not auxinic herbicide damage. These resultsindicated commercial level tolerance can be provided by AAD-1 (v3), evenin a very auxin-sensitive dicot crop like tobacco. These results alsoshow resistance can be imparted to both chiral(2,4-dichlorophenoxypropionic acid) and achiral(2,4-dichlorophenoxyacetic acid) phenoxy auxin herbicides alone or intank mix combination.

TABLE 28 Segregating AAD-1 T₁ tobacco plants' response to phenoxy auxinherbicides. 721(2)013.010 721(3)008.005 KY160 - (medium tolerance (hightolerance Wildtype in T₀ generation) in T₀ generation) Herbicide Average% Injury of Replicates 14 DAT 560 g ae/ha 2,4-D DMA 75 5 0 1120 g ae/ha2,4-D DMA 80 5 2 2240 g ae/ha 2,4-D DMA 90 5 0 4480 g ae/ha 2,4-D DMA 955 5 560 g ae/ha R-dichlorprop 70 5 0 1120 g ae/ha R-dichlorprop 75 5 02240 g ae/ha R-dichlorprop 88 5 0 4480 g ae/ha R-dichlorprop 95 10 5 560g ae/ha 2,4-D DMA/R-dichlorprop 80 5 5 1120 g ae/ha 2,4-DDMA/R-dichlorprop 80 10 10 2240 g ae/ha 2,4-D DMA/R-dichlorprop 95 15 154480 g ae/ha 2,4-D DMA/R-dichlorprop 95 15 15

A 100 plant progeny test was also conducted on each of the two AAD-1(v3) lines (events 721(2)-013.010 and 721(3)-008.005). The seeds werestratified, sown, and transplanted with respect to the procedure aboveexcept null plants were not removed by Liberty selection. All plantswere then sprayed with 560 g ae/ha 2,4-D DMA as previously described.After 14 DAT, resistant and sensitive plants were counted. Both event‘013’ and ‘008’ segregated as a single locus, dominant Mendelian trait(3R:1S) as determined by Chi square analysis. AAD-1 is heritable as arobust phenoxy auxin resistance gene in multiple species.

Field level tolerance will be demonstrated by planting T₁ or T₂ seed inthe greenhouse, selectively removing the null plants by Libertyselection as previously described, and rearing individual seedlings in72-well transplant flats (Hummert International) in Metro 360 mediaaccording to growing conditions indicated above. Individual plants willbe transplanted into the field plots using an industrial vegetableplanter. Drip or overhead irrigation will be used to keep plants growingvigorously. Once plants reach 6-12 inches in height, tobacco plants willbe sprayed with a broad range of phenoxy auxins and rated as shownabove. Environmental stresses are more significant under fieldconditions; however, based on previous experience with AAD-1(v3)-transformed corn, robust translation of resistance from thegreenhouse to the field is expected.

Example 13—Soybean Transformation

Soybean improvement via gene transfer techniques has been accomplishedfor such traits as herbicide tolerance (Padgette et al., 1995), aminoacid modification (Falco et al., 1995), and insect resistance (Parrottet al., 1994). Introduction of foreign traits into crop species requiresmethods that will allow for routine production of transgenic lines usingselectable marker sequences, containing simple inserts. The transgenesshould be inherited as a single functional locus in order to simplifybreeding. Delivery of foreign genes into cultivated soybean bymicroprojectile bombardment of zygotic embryo axes (McCabe et al., 1988)or somatic embryogenic cultures (Finer and McMullen, 1991), andAgrobacterium-mediated transformation of cotyledonary explants (Hincheeet al., 1988) or zygotic embryos (Chee et al., 1989) have been reported.

Transformants derived from Agrobacterium-mediated transformations tendto possess simple inserts with low copy number (Birch, 1991). There arebenefits and disadvantages associated with each of the three targettissues investigated for gene transfer into soybean, zygotic embryonicaxis (Chee et al., 1989; McCabe et al., 1988), cotyledon (Hinchee etal., 1988) and somatic embryogenic cultures (Finer and McMullen, 1991).The latter have been extensively investigated as a target tissue fordirect gene transfer. Embryogenic cultures tend to be quite prolific andcan be maintained over a prolonged period. However, sterility andchromosomal aberrations of the primary transformants have beenassociated with age of the embryogenic suspensions (Singh et al., 1998)and thus continuous initiation of new cultures appears to be necessaryfor soybean transformation systems utilizing this tissue. This systemneeds a high level of 2,4-D, 40 mg/L concentration, to initiate theembryogenic callus and this poses a fundamental problem in using theAAD-1 (v3) gene since the transformed locus could not be developedfurther with 2,4-D in the medium. So, the meristem based transformationis ideal for the development of 2,4-D resistant plant using AAD-1 (v3).

13.1—Transformation Method 1: Cotyledonary Node Transformation ofSoybean Mediated by Agrobacterium tumefaciens.

The first reports of soybean transformation targeted meristematic cellsin the cotyledonary node region (Hinchee et al., 1988) and shootmultiplication from apical meristems (McCabe et al., 1988). In the A.tumefaciens-based cotyledonary node method, explant preparation andculture media composition stimulate proliferation of auxiliary meristemsin the node (Hinchee et al., 1988). It remains unclear whether a trulydedifferentiated, but totipotent, callus culture is initiated by thesetreatments. The recovery of multiple clones of a transformation eventfrom a single explant and the infrequent recovery of chimeric plants(Clemente et al., 2000; Olhoft et al., 2003) indicates a single cellorigin followed by multiplication of the transgenic cell to produceeither a proliferating transgenic meristem culture or a uniformlytransformed shoot that undergoes further shoot multiplication. Thesoybean shoot multiplication method, originally based on microprojectilebombardment (McCabe et al., 1988) and, more recently, adapted forAgrobacterium-mediated transformation (Martinell et al., 2002),apparently does not undergo the same level or type of dedifferentiationas the cotyledonary node method because the system is based onsuccessful identification of germ line chimeras. The range of genotypesthat have been transformed via the Agrobacterium-based cotyledonary nodemethod is steadily growing (Olhoft and Somers, 2001). This de novomeristem and shoot multiplication method is less limited to specificgenotypes. Also, this is a non 2,4-D based protocol which would be idealfor 2,4-D selection system. Thus, the cotyledonary node method may bethe method of choice to develop 2,4-D resistant soybean cultivars.Though this method was described as early as 1988 (Hinchee et al.,1988), only very recently has it been optimized for routine highfrequency transformation of several soybean genotypes (Zhang et al.,1999; Zeng et al., 2004).

13.1.1—Agrobacterium preparation. The plasmid, pDAB721, contains theAAD-1 (v3) gene under the control of the Arabidopsis Ubi10 promoter.This plasmid also carries the PAT gene under the control of rice actinpromoter coding for an enzyme that degrades glufosinate which can beused as a selection agent for transformants. This vector can be used inthe transformation experiment described below. The construct pDAB721 wasmobilized into the Agrobacterium strain EHA101S by electroporation.

Agrobacterium cultures harboring pDAB721 used in the transformations canbe grown in YEP medium (10 g/L peptone, 5 g/L yeast extract and 5 g/LNaCl, pH 7.0). Agrobacterium cultures are pelleted at low speed andresuspended in SCM liquid medium (see below) to OD660 of 0.6 for use inthe inoculations.

13.1.2—Plant transformation. Seeds of “Thorne,” “Williams82,” or“NE3001,” public genotypes of soybean, can be disinfected by a 20-minutewash in 20% (v/v) commercial bleach (NaClO) amended with 2 drops ofLiqui-Nox®. The seeds should be rinsed five times with sterile water onhormone-free SHGA medium and allowed to germinate for 5 days at 24° C.,with an 18/6 hour light/dark regime. B5 medium consists of macro andmicro nutrients and vitamins described by Gamborg et al. (1968) (Sigma,cat. #G 5893, St. Louis). All media are solidified with 0.8% (w/v)washed agar (Sigma cat. #A 8678). Alternatively, certain genotypes ofsoybean can undergo a dry surface sterilization procedure using chlorinegas (Cl₂) whereby mature seeds are placed into 100×25 mm Petri plates ina single layer using about 130 seeds per plate. Approximately 3-4 platesare placed into a desiccator within a fume hood in such a way that allthe plates are half open and that there is enough space for a 250 mlbeaker in the middle of the desiccator. The beaker is filled with 95 mlof commercial bleach, to which 5 ml of concentrated (12N) HCl is addeddropwise along the side of the beaker. The desiccator is immediatelyclosed and allowed to stand for at least 16 hours in a fume hood.Following sterilization, the plates are closed, then brought to alaminar flow hood and left open for about 30 minutes to remove anyexcessive Cl₂ gas. Seeds are then germinated as described previously.The dry surface sterilized seeds will remain sterile at room temperaturefor about 2 weeks. Explants are prepared for inoculation as previouslydescribed (Hinchee et al., 1988).

Explants should be inoculated for 30 minutes. Cocultivation andAgrobacterium re-suspension medium consists of B5 medium supplementedwith 1 mg/L BAP, 1 mg/L GA3, 3% (w/v) sucrose, 20 mM MES(2-1N-morpholinolethane sulfonic acid), 400 mg/L L-cysteine (Olhoft andSomers, 2001) pH 5.7, 0.99 mM dithiothreitol (DTT), and 200 μMacetosyringone (AS). Explants are cocultivated for 5 days at 25° C.Following cocultivation, explants were washed in the cocultivationmedium containing 100 mg/L timentin and 200 mg/L cefotaxime, without MESand AS.

Explants are to be placed on shoot induction medium and transferredevery 14 days for a total of 28 days prior to herbicide selection. Theshoot induction medium consists of full-strength B5 medium supplementedwith 1.7 mg/L BAP, 100 mg/L timentin, 200 mg/L cefotaxime pH 5.7 and 3%(w/v) sucrose. Cotyledons should be placed adaxial side up with thecotyledonary nodal region flush to the medium, amended with increasinglevels of Basta (2, 5, 6 then 7 mg/L glufosinate ammonium) or sublethallevels of 2,4-D ranging from 10 mg to 400 mg/L every 2 weeks for a totalof 8 weeks.

Differentiating explants are subsequently transferred to shootelongation medium for an additional 4 to 10 weeks under the sameglufosinate selection or under decreased 2,4-D selection pressureranging from 10 mg to 100 mg/L. The elongation medium consisted of B5medium (Sigma cat. #M0404) amended with 1 mg/L zeatin riboside, 0.1 mg/LIAA (indole-3-acetic acid), 0.5 mg/L GA3, 50 mg/L glutamine, 50 mg/Lasparagine, and 3% (w/v) sucrose, pH 5.8. Elongated shoots should berooted, without further selection, on half-strength MS/B5 medium withfull-strength vitamins plus 0.5 mg/L NAA (α-naphthaleneacetic acid) or0.1 mg/L IAA and 2% (w/v) sucrose.

The antibiotics, timentin and cefotaxime, are maintained within themedia throughout selection. Cultures are transferred to fresh mediumevery 2 weeks. Plantlets are acclimated for 1 to 2 weeks prior totransfer to the greenhouse.

13.1.3—Progeny evaluation. To plants will be allowed to self fertilizein the greenhouse to give rise to T₁ seed. T₁ plants (and to the extentenough T₀ plant clones are produced) will be sprayed with a range ofherbicide doses to determine the level of herbicide protection affordedby AAD-1 (v3) and PAT genes in transgenic soybean. Rates of 2,4-D usedon T₀ plants will typically use one or two selective rates in the rangeof 100-400 g ae/ha. T₁ seed will be treated with a wider herbicide doseranging from 50-3200 g ae/ha 2,4-D. Likewise, T₀ and T₁ plants can bescreened for glufosinate resistance by postemergence treatment with200-800 and 50-3200 g ae/ha glufosinate, respectively. Analysis ofprotein expression will occur as described in Example 9 for Arabidopsisand corn. Determination of the inheritance of AAD-1 (v3) will be madeusing T₁ and T₂ progeny segregation with respect to herbicide tolerance.

13.2—Transformation Method 2: “No-Shake” Agrobacterium MediatedTrans-Formation of Non-Regenerable Soybean Suspension Cells.

The DAS Soybean cell suspensions were cultured on a 3d cycle with 10 mlof settled suspension volume transferred to 115 ml of fresh liquidmedium. Settled cell volume was determined by allowing the cellsuspension to settle for 2 min in the 125-mL flask after vigorousswirling and then drawing cells from the bottom of the flask with a widebore 10 ml pipette. The flasks were then transferred to orbital shakersat 140 rpm.

Aliquots of 4 ml of the suspension cells at 0.72 OD⁶⁰⁰ was transferredalong with 200 μM acetosyringone (AS) onto a 100×25 sterile Petri plate.EHA105 Agrobacterium suspension at a density of 1.2 OD⁶⁵⁰ in a 100 μLvolume was added and mixed well. The Agrobacterium and suspension cellmixture was swirled well and the plate was transferred to dark growthchamber where the temperature was maintained at 25° C.

13.2.1—Selection of Soybean Suspension Cells and Isolation ofTransformed Colonies.

After 4 days of co-cultivation the plate was swirled again to mix thesuspension well and a 1.5 ml aliquot was transferred to the selectionmedium and spread on the gel medium on a 100×15 ml Petri-plate. Theselection medium consisted of full-strength B5 medium supplemented with1.7 mg/L BAP, 100 mg/L timentin, 200 mg/L cefotaxime pH 5.7 and 3% (w/v)sucrose and the medium was amended with glufosinate ammonium at 5 mg/Llevel. After a 10 min drying in the hood the plates were incubated for 4weeks in dark at 28° C. Colonies appeared in selection and a total of 11colonies were transferred to fresh medium from 3 different experimentsand maintained for 3-4 months. All the 11 resistant colonies producedcalli that were growing on the 5 mg/L glufosinate containing selectionmedium. The non-transformed suspension cells were sensitive when platedon to 0.5 mg/l glufosinate ammonium medium. However, the transformedcolonies were resistant to 5× concentration of glufosinate ammonium andwere maintained for up to 4 months.

Callus events were sampled for analyses when they reached 2 to 3 cm indiameter. At least two of colony isolates, one each from two differentexperiments, were analyzed for AAD1 protein expression. The ELISA andWestern analysis carried out on these two isolates showed positiveexpression of AAD1 proteins. Both ELISA (Table 29) and Western Blotting(FIG. 18) analysis on two separate soybean calli transformed with AAD-1(v3) gene indicated that the callus cells are expressing AAD-1 (v3)protein. The sandwich ELISA detected 0.0318% and 0.0102% total solubleprotein of AAD-1 (v3) in two different callus tissue samples. Due to thesensitivity and cross reactivity of the antiserum, multiple bands wereobserved in the western blot. However, AAD-1 (v3) specific band wasobserved in both callus samples but not in the wild type (negative)tissue. Coding region PCR analyses showed the expected size products ofthe AAD1 and the PAT coding regions in these colonies indicating thatthey were transformed.

TABLE 29 PCR and ELISA data for transgenic soybean events. AAD-1 PATCoding Coding TSP AAD-1 % Event region PCR region PCR (μg/mL) (ng/mL)Expression 1-1 + + 1995.13 633.89 0.0318% 2-1 + + 2018.91 205.92 0.0102%Negative − − 2074.63 −1.22 −0.0001% Control

13.3—Transformation Method 3: Aerosol-Beam Mediated Transformation ofEmbryogenic Soybean Callus Tissue.

Culture of embryogenic soybean callus tissue and subsequent beaming wereas described in U.S. Pat. No. 6,809,232 (Held et al.).

Embryogenic calluses of several Stine elite varieties, including 96E750,96E94, 97E986, 96E144 and 96E692, were separately collected into thecenter of plates of B1-30 3Co5My or B1-30 3Co5My0.25PA0.5K three daysafter transfer to fresh medium. The tissue was then beamed with pDAB3295using linearized DNA at a concentration of approximately 0.2 μg/ml.After beaming, the embryogenic callus was transferred to fresh B1-303Co5My or B1-30 3Co5My0.25PA0.5K for one passage of a month. The tissuewas then transferred to selective medium containing 1 mg/l bialaphos.With bialaphos, selection typically was maintained at 1 mg/l for thefirst two one-month passages and then increased to 2 mg/l for thefollowing three to seven months. Transgenic events were identified whencallus tissue generated by transformation experiments began to organizeand develop into embryogenic structures while still on selective mediacontaining 2,4-D plus bialaphos. Once identified, the maturingstructures were regenerated into plants according to the followingprotocol: Embryogenic structures were transferred off B1-30 3Co5My orB1-30 3/co5My0.25PA0.5K to B3 medium. After 3 to 4 weeks' growth on B3medium, individual structures were transferred to fresh medium. Afteranother 3 to 4 weeks, maturing embryos were transferred to B5G mediumand placed in the light. Embryos that had elongated and produced rootswere transferred to tubes containing ½ B5G medium where they continueddevelopment into plantlets; and these plantlets were removed from thetubes and placed into pots.

Variations of media referred to in Table 30 were tested, e.g., B1-303Co5My, which was made by adding 3% coconut water and 5 gm/lmyo-inositol to B1-30. Other variations included: B1-30 3Co5My0.25PA0.5K which contained B1-30 basal medium plus 3% coconut water, 5 gm/lmyo-inositol, 0.25 gm/l phytic acid, and 0.5 gm/l additional KH₂PO₄ and½ B5G which contained all ingredients of B5G medium at half strength.

TABLE 30 Growth Media for Soybean Ingredients in 1 Liter B1-30 B3 B5G MsSalts 4.43 g 4.43 g B5 Salts 3.19 g NaEDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D30 mg Activated charcoal 5 g Phytagar 8 g 8 g Gelrite 2 g pH 5.8 5.8 5.8

Example 14—AAD-1 (v3) Enablement in Cotton

14.1—Cotton Transformation Protocol.

Cotton seeds (Co310 genotype) are surface-sterilized in 95% ethanol for1 minute, rinsed, sterilized with 50% commercial bleach for twentyminutes, and then rinsed 3 times with sterile distilled water beforebeing germinated on G-media (Table 31) in Magenta GA-7 vessels andmaintained under high light intensity of 40-60 μE/m2, with thephotoperiod set at 16 hours of light and 8 hours dark at 28° C.

Cotyledon segments (˜5 mm) square are isolated from 7-10 day oldseedlings into liquid M liquid media (Table 31) in Petri plates (Nunc,item #0875728). Cut segments are treated with an Agrobacterium solution(for 30 minutes) then transferred to semi-solid M-media (Table 31) andundergo co-cultivation for 2-3 days. Following co-cultivation, segmentsare transferred to MG media (Table 31). Carbenicillin is the antibioticused to kill the Agrobacterium and glufosinate-ammonium is the selectionagent that would allow growth of only those cells that contain thetransferred gene.

Agrobacterium preparation. Inoculate 35 ml of Y media (Table 31)(containing streptomycin (100 mg/ml stock) and erythromycin (100 mg/mlstock)), with one loop of bacteria to grow overnight in the dark at 28°C., while shaking at 150 rpm. The next day, pour the Agrobacteriumsolution into a sterile oakridge tube (Nalge-Nunc, 3139-0050), andcentrifuge for in Beckman J2-21 at 8,000 rpm for 5 minutes. Pour off thesupernatant and resuspend the pellet in 25 ml of M liquid (Table 31) andvortex. Place an aliquot into a glass culture tube (Fisher, 14-961-27)for Klett reading (Klett-Summerson, model 800-3). Dilute the newsuspension using M liquid media to a Klett-meter reading of 10⁸ colonyforming units per mL with a total volume of 40 ml.

After three weeks, callus from the cotyledon segments is isolated andtransferred to fresh MG media. The callus is transferred for anadditional 3 weeks on MG media. Callus is then transferred to CG-media(Table 31), and transferred again to fresh selection medium after threeweeks. After another three weeks the callus tissue is transferred to Dmedia (Table 31) lacking plant growth regulators for embryogenic callusinduction. After 4-8 weeks on this media, embryogenic callus is formed,and can be distinguished from the non-embryogenic callus by itsyellowish-white color and granular cells. Embryos start to regeneratesoon after and are distinct green in color.

Larger, well-developed embryos are isolated and transferred to DK media(Table 31) for embryo development. After 3 weeks (or when the embryoshave developed), germinated embryos are transferred to fresh media forshoot and root development. After 4-8 weeks, any well-developed plantsare transferred into soil and grown to maturity. Following a couple ofmonths, the plant has grown to a point that it can be sprayed todetermine if it has resistance to 2,4-D.

TABLE 31 Media for Cotton Transformation Ingredients in 1 liter G Mliquid M MG CG D DK Y LS Salts (5X) 200 ml 200 ml 200 ml 200 ml 200 mlGlucose 30 grams 30 grams 30 grams 30 grams 20 grams modified B5 vit(1000x) 1 ml 1 ml 1 ml 1 ml 1 ml 10 ml 1 ml kinetin (1 mM) 1 ml 1 ml 1ml 4.6 ml 0.5 ml 2,4-D (1 mM) 1 ml 1 ml 1 ml agar 8 grams 8 grams 8grams 8 grams 8 grams 8 grams DKW salts (D190) 1 package 1 packageMYO-Inositol (100x) 1 ml 10 ml Sucrose 3% 30 grams 30 grams 10 grams NAACarbenicillin 2 ml 0.4 ml (250 mg/ml) GLA (10 mg/ml) 0.5 ml 0.3 mlPeptone 10 grams Yeast Extract 10 grams NaCl  5 grams

14.2—Experiment Specifics.

For this experiment 500 cotyledon segments were treated with pDAB721. Ofthe 500 segments treated, 475 had callus isolated while on selection(95% transformation frequency). The callus was selected onglufosinate-ammonium, due to the inclusion of the PAT gene in theconstruct, since there was already a selection scheme developed. Callusline analysis in the form of PCR and Invader were initiated to determinethe insertion patterns and to be sure the gene was present at the callusstage, then callus lines that were embryogenic were sent for Westernanalysis.

14.3—Callus Analysis Results.

The object of the analysis is to eliminate any lines that do not havethe complete PTU, show no expression or that have high copy number, sothose lines are not regenerated. Of the 475 pDAB721 transformed calluslines, 306 were sent for PCR analysis and Invader assay (Table 32). Veryfew lines were PCR negative. The Invader results are not complete atthis time, because some samples had low DNA amounts when extracted, andthese have been resubmitted. However, the current Invader data shows afew of the lines submitted have high copy number (copy number of >2)(Table 32). Due to the large number of lines that passed analysis, itwas necessary to decrease the number of embryogenic callus lines beingmaintained due to the volume. Ninety lines have been sent for Westernanalysis, and eight of those were negative. The western analysis showedhigh expression from the majority of the lines (Table 32). Eighty-twoembryogenic callus lines are being maintained for plant regenerationbased on analysis results (and results pending).

TABLE 32 Analysis of cotton callus Line Copy # PTU Western 1 2 pos *** 21 pos *** 3 1 pos *** 4 2 pos *** 5 1 pos *** 6 1 pos neg 7 1 pos *****8 1 pos ***** 9 2 pos * 10 1 pos * 11 1 pos ***** 12 1 pos * 13 1 pos *14 1 pos ** 15 1 pos **** 16 1 pos ***** 17 1 pos * 18 1 pos *** 19 1pos ** 20 1 pos ***** 21 2 pos ***** 22 1 pos ***** 23 1 pos ***** 24 4pos * or neg 25 1 pos **** 26 1 pos **** 27 low DNA pos ***** 28 low DNApos ** 29 low DNA pos ***** 30 17  pos * 31 low DNA pos ***** 32 low DNApos ***** 33 low DNA pos **** 34 low DNA faint pos ***** 35 low DNA pos**** 36 low DNA pos neg 37 low DNA pos **** 38 low DNA neg ***** 39 1pos **** 40 low DNA pos * 41 low DNA pos * 42 low DNA pos ** standardAAD1  5 ug/ml ***** standard AAD1 0.5 ug/ml ** 43 1 pos ***** 44 low DNApos ***** 45 1 pos ***** 46 2 pos ***** 47 1 pos *** 48 1 pos *** 49 3faint pos neg 50 1 pos **** 51 4 pos neg 52 2 pos **** 53 1 pos *** 54 1pos **** 55 1 pos neg 56 5 pos * 57 2 faint pos neg 58 8 pos **** 59 2pos **** 60 5 pos ***** 61 1 pos ** 62 1 pos ** 63 1 pos *** 64 1 pos*** 65 3 pos **** 66 5 pos * 67 6 pos * 68 Low DNA pos neg 69 Low DNApos **** 70 low DNA pos ** 71 low DNA faint pos ** 72 low DNA pos ****73 low DNA neg *** 74 low DNA faint pos *** 75 Low DNA pos *** 76 LowDNA pos neg 77 low DNA faint pos **** 78 low DNA pos **** 79 1 pos ** 80low DNA pos *** 81 low DNA pos *** 82 low DNA pos *** 83 low DNA neg**** 84 low DNA pos *** 85 low DNA pos ** 86 low DNA pos *

14.4—Plant Regeneration.

Two AAD-1 (v3) cotton lines have produced plants according to the aboveprotocol that have been sent to the greenhouse. To demonstrate the AAD-1(v3) gene provides resistance to 2,4-D in cotton, both the AAD-1 (v3)cotton plant and wild-type cotton plants were sprayed with a tracksprayer set at 187 L/ha. The plants were sprayed at 560 g ae/ha 2,4-DMAformulated in 200 mM Hepes buffer (pH 7.5). The plants were evaluated at3, 7, and 14 days after treatment. Plants were assigned injury ratingswith respect to stunting, chlorosis, and necrosis. Plants assigned aninjury rating of 90% or above are considered dead. Three days aftertreatment (DAT) the wild-type plant began showing epinasy and received arating of 15%; in contrast, the AAD-1 (v3) plant showed 0% injury. By 7DAT epinasy continued on the wild-type and the new growth shoots beganturning brown. It received a rating of 50% at this time. At 14 DAT theAAD-1 (v3) plant was still uninjured, whereas the wild-type was severelystunted, and the new growth areas were brown and shriveled. Thus, thewild-type received a rating of 90% at 14 DAT.

This study demonstrates that the AAD-1 (v3) gene in cotton providessubstantial tolerance to 2,4-D up to at least 560 g ae/ha.

Example 15—Agrobacterium Transformation of Other Crops

In light of the subject disclosure, additional crops can be transformedaccording to the subject invention using techniques that are known inthe art. For Agrobacterium-mediated trans-formation of rye, see, e.g.,Popelka and Altpeter (2003). For Agrobacterium-mediated transformationof soybean, see, e.g., Hinchee et al., 1988. For Agrobacterium-mediatedtransformation of sorghum, see, e.g., Zhao et al., 2000. ForAgrobacterium-mediated transformation of barley, see, e.g., Tingay etal., 1997. For Agrobacterium-mediated transformation of wheat, see,e.g., Cheng et al., 1997. For Agrobacterium-mediated transformation ofrice, see, e.g., Hiei et al., 1997.

The Latin names for these and other plants are given below. It should beclear that these and other (non-Agrobacterium) transformation techniquescan be used to transform AAD-1 (v3), for example, into these and otherplants, including but not limited to Maize (Gramineae Zea mays), Wheat(Pooideae Triticum spp.), Rice (Gramineae Oryza spp. and Zizania spp.),Barley (Pooideae Hordeum spp.), Cotton (Abroma Dicotyledoneae Abromaaugusta, and Malvaceae Gossypium spp.), Soybean (Soya LeguminosaeGlycine max), Sugar beet (Chenopodiaceae Beta vulgaris altissima), Sugarcane (Arenga pinnata), Tomato (Solanaceae Lycopersicon esculentum andother spp., Physalis ixocarpa, Solanum incanum and other spp., andCyphomandra betacea), Potato, Sweet potato, Rye (Pooideae Secale spp.),Peppers (Solanaceae Capsicum annuum, sinense, and frutescens), Lettuce(Compositae Lactuca sativa, perennis, and pulchella), Cabbage, Celery(Umbelliferae Apium graveolens), Eggplant (Solanaceae Solanummelongena), Sorghum (all Sorghum species), Alfalfa (Leguminosae Medicagosativum), Carrot (Umbelliferae Daucus carota sativa), Beans (LeguminosaePhaseolus spp. and other genera), Oats (Avena Sativa and Strigosa), Peas(Leguminosae Pisum, Vigna, and Tetragonolobus spp.), Sunflower(Compositae Helianthus annuus), Squash (Dicotyledoneae Cucurbita spp.),Cucumber (Dicotyledoneae genera), Tobacco (Solanaceae Nicotiana spp.),Arabidopsis (Cruciferae Arabidopsis thaliana), Turfgrass (Lolium,Agrostis, and other families), and Clover (Leguminosae). Such plants,with AAD-1 (v3) genes, for example, are included in the subjectinvention.

Example 16—Stacking AAD-1 (v3) with AHAS Herbicide Resistance Gene

Stacking AAD-1 (v3) with an AHAS herbicide resistance gene is describedin Example 7.9.

Example 17—Further Evidence of Surprising Results: AAD-1 vs. AAD-2

17.1—AAD-2 (v1) Initial Cloning.

Another gene was identified from the NCBI database (see the ncbi.nlmnih.gov website; accession #AP005940) as a homologue with only 44% aminoacid identity to tfdA. This gene is referred to herein as AAD-2 (v1) forconsistency. Percent identity was determined by first translating boththe AAD-2 and tfdA DNA sequences (SEQ ID NO:12 and GENBANK Accession No.M16730, respectively) to proteins (SEQ ID NO:13 and GENBANK AccessionNo. M16730, respectively), then using ClustalW in the VectorNTI softwarepackage to perform the multiple sequence alignment.

The strain of Bradyrhizobium japonicum containing the AAD-2 (v1) genewas obtained from Northern Regional Research Laboratory (NRRL, strain#B4450). The lyophilized strain was revived according to NRRL protocoland stored at −80° C. in 20% glycerol for internal use as Dow Bacterialstrain DB 663. From this freezer stock, a plate of Tryptic Soy Agar wasthen struck out with a loopful of cells for isolation, and incubated at28° C. for 3 days. A single colony was used to inoculate 100 ml ofTryptic Soy Broth in a 500 ml tri-baffled flask, which was incubatedovernight at 28° C. on a floor shaker at 150 rpm. From this, total DNAwas isolated with the gram negative protocol of Qiagen's DNeasy kit(Qiagen cat. #69504). The following primers were designed to amplify thetarget gene from genomic DNA, Forward: 5′ ACT AGT AAC AAA GAA GGA GATATA CCA TGA CGA T 3′ [(brjap 5′(spel) SEQ ID NO:14 (added Spe Irestriction site and Ribosome Binding Site (RBS))] and Reverse: 5′ TTCTCG AGC TAT CAC TCC GCC GCC TGC TGC TGC 3′ [(br jap 3′ (xholI) SEQ IDNO:15 (added a Xho I site)].

Fifty microliter reactions were set up as follows: Fail Safe Buffer 25μl, ea. primer 1 μl (50 ng/μl), gDNA 1 μl (200 ng/μl), H₂O 21 μl, Taqpolymerase 1 μl (2.5 units/μl). Three Fail Safe Buffers—A, B, and C—wereused in three separate reactions. PCR was then carried out under thefollowing conditions: 95° C. 3.0 minutes heat denature cycle; 95° C. 1.0minute, 50° C. 1.0 minute, 72° C. 1.5 minutes, for 30 cycles; followedby a final cycle of 72° C. 5 minutes, using the FailSafe PCR System(Epicenter cat. #FS99100). The resulting ˜1 kb PCR product was clonedinto pCR 2.1 (Invitrogen cat. #K4550-40) following the includedprotocol, with chemically competent TOP10F′ E. coli as the host strain,for verification of nucleotide sequence.

Ten of the resulting white colonies were picked into 3 μl LuriaBroth+1000 μg/ml Ampicillin (LB Amp), and grown overnight at 37° C. withagitation. Plasmids were purified from each culture using NucleospinPlus Plasmid Miniprep Kit (BD Biosciences cat. #K3063-2) and followingincluded protocol. Restriction digestion of the isolated DNA's wascompleted to confirm the presence of the PCR product in the pCR2.1vector. Plasmid DNA was digested with the restriction enzyme EcoRI (NewEngland Biolabs cat. #R0101S). Sequencing was carried out with BeckmanCEQ Quick Start Kit (Beckman Coulter cat. #608120) using M13 Forward [5′GTA AAA CGA CGG CCA GT 3] (SEQ ID NO:16) and Reverse [5′ CAG GAA ACA GCTATG AC 3] (SEQ ID NO:17) primers, per manufacturers instructions. Thisgene sequence and its corresponding protein was given a new generaldesignation AAD-2 (v1) for internal consistency.

17.2—Completion of AAD-2 (v1) Binary Vector.

The AAD-2 (v1) gene was PCR amplified from pDAB3202. During the PCRreaction alterations were made within the primers to introduce theAflIII and SacI restriction sites in the 5′ primer and 3′ primer,respectively. The primers “NcoI of Brady” [5′ TAT ACC ACA TGT CGA TCGCCA TCC GGC AGC TT 3] (SEQ ID NO:18) and “SacI of Brady” [5′ GAG CTC CTATCA CTC CGC CGC CTG CTG CTG CAC 3] (SEQ ID NO:19) were used to amplify aDNA fragment using the Fail Safe PCR System (Epicentre). The PCR productwas cloned into the pCR 2.1 TOPO TA cloning vector (Invitrogen) andsequence verified with M13 Forward and M13 Reverse primers using theBeckman Coulter “Dye Terminator Cycle Sequencing with Quick Start Kit”sequencing reagents. Sequence data identified a clone with the correctsequence (pDAB716). The AflIII/SacI AAD-2 (v1) gene fragment was thencloned into the NcoI/SacI pDAB726 vector. The resulting construct(pDAB717); AtUbi10 promoter: Nt OSM 5′UTR: AAD-2 (v1): Nt OSM3′UTR: ORF1polyA 3′UTR was verified with restriction digests (with NcoI/SacI). Thisconstruct was cloned into the binary pDAB3038 as a NotI-NotI DNAfragment. The resulting construct (pDAB767); AtUbi10 promoter: NtOSM5′UTR: AAD-2 (v1): Nt OSM 3′UTR: ORF1 polyA 3′UTR: CsVMV promoter:PAT: ORF25/26 3′UTR was restriction digested (with NotI, EcoRI, HinDIII,NcoI, PvuII, and SalI) for verification of the correct orientation. Thecompleted construct (pDAB767) was then used for transformation intoAgrobacterium.

17.3—Comparison of Substrate Specificities of AAD-2 (v1) and AAD-1 (v1)

The activity of an extract from E. coli expressing AAD-2 (v1) (pDAB3202)prepared as in Example 11 was tested on four herbicides, 2,4-D,(R,S)-dichlorprop, (R,S)-haloxyfop and (R)-haloxyfop (all at a finalconcentration of 0.5 mM) using 3 μl (42 μg) of E. coli extract per assaywith a 15 min assay period. FIG. 22 shows that the relative AAD-2 (v1)activity on the substrates was2,4-D=dichlorprop>(R,S)-haloxyfop>>(R)-haloxyfop. Thus AAD-2 (v1)differs from AAD-1 (v1) in that it has a similar level of activity on2,4-D as on dichlorprop (whereas the activity of AAD-1 (v1) on 2,4-D is˜10% that of dichlorprop). AAD-2 (v1) also differs from AAD-1 (v1) inthat it is unable to act on (R)-haloxyfop. Table 33 shows data fromadditional substrates tested with AAD-1 (v1) and AAD-2 (v1) that confirmthat AAD-2 (v1) is specific for (S)-enantiomer substrates, in contrastto AAD-1 (v1) which is specific for (R)-enantiomers. In another test,AAD-2 (v1) was found to differ from AAD-1 (v1) in that it releaseslittle or no detectable phenol from 2,4-D sulfonate (in which asulfonate group replaces the carboxylate of 2,4-D) whereas AAD-1 (v1)produces significant levels of phenol from this compound (˜25% of2,4-D.)

TABLE 33 Comparison of AAD1 and AAD2 activity on various substrates.Substrates were assayed at 0.5 mM for 15 min in 25 mM MOPS pH 6.8, 200μM Fe²⁺, 200 μM Na ascorbate, 1 mM α- ketoglutarate using 4 μl AAD1 (32μg protein) extract or 3 μl AAD2 extract (42 μg protein). A510 STRUCTUREReg ID Compound Enantiomer AAD1 AAD2

−8706 quizalofop R 0.27 0.01

8671 haloxyfop R 0.12 0

66905 haloxyfop R,S 0.1 0.3

−4623 cyhalofop R,S 0.12 0.1

−4603 cyhalofop R 0.14 0

7466 cyhalofop S 0 0.15

11044492 fenoxaprop R 0.14 0

43865 haloxyfop-acetate — 0 0.22

The enzyme kinetics of partially purified AAD-1 (v1) and AAD-2 (v1) werecompared using 2,4-D as substrate. See FIG. 19. The K_(m) values for2,4-D were 97 and 423 μM for AAD-1 (v1) and AAD-2 (v1) respectively andthe apparent V_(max) values were 0.11 and 0.86 A₅₁₀ units, respectively(Table 34). Because equivalent amounts of enzyme were used in the assays(as determined by SDS-PAGE analysis), it can be concluded that thek_(cat) of AAD-2 (v1) for 2,4-D is almost 8-fold higher than AAD-1 (v1)and the k_(cat)/K_(m) is 2-fold higher. Thus AAD-2 (v1) is significantlymore efficient at cleaving 2,4-D in vitro than AAD-1 (v1). This is insurprising contrast to the in planta findings reported below, whereplants expressing AAD-1 (v1) are significantly better in conferring2,4-D resistance relative to AAD-2 (v1).

TABLE 34 Comparison of the Km and “Vmax” values for the aryloxyalkanoatedioxygenases (AADs) from pDAB3202 (AAD-2) and pDAB3203 [AAD-1 (v1)] withdifferent herbicide substrates: Km (μM) ± Vmax Km/Vmax Enzyme CompoundSE (A510 units) (arbitrary units) AAD-2 2,4-D 423 (±1) 0.86 2.03 AAD-1(v1) 2,4-D  97 (±21) 0.11 1.16 Notes: Assays were performed in MOPS pH6.75 + 1 mM α-ketoglutarate + 0.1 mM Na ascorbate + 0.1 mM Fe2+ and thereleased phenols colorimetrically detected using4-aminoantipyrine/ferricyanide.

17.4—Evaluation of Transformed Arabidopsis.

Freshly harvested T₁ seed transformed with a native [AAD-1 (v2)], plantoptimized [AAD-1 (v3)], or native AAD-2 (v1) gene was allowed to dry for7 days at room temperature. T₁ seed was sown in 26.5×51-cm germinationtrays (T.O. Plastics Inc., Clearwater, Minn.), each receiving a 200 mgaliquots of stratified T₁ seed (˜10,000 seed) that had previously beensuspended in 40 ml of 0.1% agarose solution and stored at 4° C. for 2days to complete dormancy requirements and ensure synchronous seedgermination.

Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) wascovered with fine vermiculite and subirrigated with Hoagland's solutionuntil wet and then allowed to gravity drain. Each 40 ml aliquot ofstratified seed was sown evenly onto the vermiculite with a pipette andcovered with humidity domes (KORD Products, Bramalea, Ontario, Canada)for 4-5 days. Domes were removed 1 day prior to initial transformantselection using glufosinate postemergence spray (selecting for theco-transformed PAT gene).

Five to six days after planting (DAP) and again 10 DAP, T₁ plants(cotyledon and 2-4-1f stage, respectively) were sprayed with a 0.2%solution of Liberty herbicide (200 g ai/L glufosinate, Bayer CropSciences, Kansas City, Mo.) at a spray volume of 10 ml/tray (703 L/ha)using a DeVilbiss compressed air spray tip to deliver an effective rateof 280 g ai/ha glufosinate per application. Survivors (plants activelygrowing) were identified 5-7 days after the final spraying andtransplanted individually into 3-inch pots prepared with potting media(Metro Mix 360). Transplanted plants were covered with humidity domesfor 3-4 days and placed in a 22° C. growth chamber as before. Domes weresubsequently removed and plants moved to the greenhouse (22±5° C.,50±30% RH, 14 hours light:10 dark, minimum 500 μE/m²s¹natural+supplemental light) at least 1 day prior to testing for theability of AAD-1 (v3), AAD-1 (v2), or AAD-2 (v1) to provide phenoxyauxin herbicide resistance.

Random individual T₁ plants selected for glufosinate resistance abovewere confirmed for expression of the PAT protein using a PAT ELISA kit(Part no. 7000045, Strategic Diagnostics, Inc., Newark, Del.) tonon-destructively confirm fidelity of selection process (manufacturer'sprotocol). Plants were then randomly assigned to various rates of 2,4-D(50-800 g ae/ha).

Herbicide applications were applied by track sprayer in a 187 L/ha sprayvolume. 2,4-D used was the commercial dimethylamine salt formulation(456 g ae/L, NuFarm, St Joseph, Mo.) mixed in 200 mM Tris buffer (pH9.0).

17.5—Results of Selection of Transformed Plants.

The first Arabidopsis transformations were conducted using AAD-1 (v3).T₁ transformants were first selected from the background ofuntransformed seed using a glufosinate selection scheme. Over 400,000 T₁seed were screened and 493 glufosinate resistant plants were identified(PAT gene), equating to a transformation/selection frequency of 0.12%.Depending on the lot of seed tested, this ranged from 0.05-0.23% (seeTable 15 in Example 6.5 above). A small lot of native AAD-1 (v2)transformed seed were also selected using the glufosinate selectionagent. Two hundred seventy eight glufosinate-resistant T₁ individualswere identified out of 84,000 seed screened (0.33%transformation/selection frequency). Surprisingly, Arabidopsistransformations using the native AAD-2(v1) gene provided a very lowtransformation frequency when selected for glufosinate tolerance (PATselectable marker function). Approximately 1.3 million seed have beenscreened and only 228 glufosinate transformants were recovered, equatingto a transformation/selection frequency of 0.018% (see Table 15).Transformation frequency for native AAD-2 (v1) was only 6% of that ofnative AAD-1 (v2). The native AAD2 (v1) gene was subsequentlysynthetically optimized, cloned, and transformed as pDAB3705, intoArabidopsis using methods previously described (see Example 5). Theplant optimized AAD2 (v2) (SEQ ID NO:29, which encodes SEQ ID NO:30)yielded a normal T₁ Arabidopsis selection frequency using Libertyherbicide of approximately 0.11% (see Table 15).

T₁ plants selected above were subsequently transplanted to individualpots and sprayed with various rates of commercial aryloxyalkanoateherbicides. Table 16 (in Example 6.5 above) compares the response ofAAD-1 (v2), AAD-1 (v3), AAD-2 (v1) and AAD-1(v2) genes to impart 2,4-Dresistance to Arabidopsis T₁ transformants. All genes did provide somesignificant 2,4-D resistance versus the transformed and untransformedcontrol lines; however, individual constructs were widely variable intheir ability to impart 2,4-D resistance to individual T₁ Arabidopsisplants. Within a given treatment, the level of plant response variedgreatly and can be attributed to the fact each plant represents anindependent transformation event. Of important note, at each 2,4-D ratetested, there were individuals that were unaffected while some wereseverely affected. An overall population injury average by rate ispresented in Table 16 simply to demonstrate the significant differencebetween the plants transformed with AAD-1 (v2), AAD-1 (v3), AAD-2 (v1)or AAD-2 (v2) versus the wildtype or PAT/Cry1F transformed controls.

Surprisingly, AAD-2 (v1) transformants were far less resistant to 2,4-Dthan either AAD-1 (v2) or AAD-1 (v3) genes (Table 16) both from afrequency of highly tolerant plants as well as overall average injury.No plants transformed with AAD-2 (v1) survived 200 g ae/ha 2,4-Drelatively uninjured (<20% visual injury), and overall population injurywas about 83%. Conversely, 56% (45 of 80) AAD-1 (v2)-transformed T₁plants survived 200 g ae/ha 2,4-D uninjured (population injuryaverage=34%), and >73% (11 of 15) AAD-1 (v3) T₁ plants were uninjured(population injury average=14%). See FIG. 20. Tolerance improvedslightly for plant-optimized AAD2 (v2) versus the native gene; however,comparison of both AAD-1 and -2 plant optimized genes indicates asignificant advantage for AAD-1 (v3) in planta (see Table 16).

These results are unexpected given that in vitro comparison of nativeAAD-1 (v2) and AAD-2 (v1) indicated 2,4-D was better degraded by AAD-2(v1). AAD-2 (v1) is expressed in individual T₁ plants to varying levelswith the anticipated size; however, little protection from 2,4-D injuryis afforded by this expressed protein. Little correlation exists betweenexpression level noted on the Western blot and the level of injury from2,4-D on the same plants. See FIG. 21. No large difference was evidentin protein expression level (in planta) for the native and plantoptimized AAD-2 genes. These data corroborate earlier findings that makethe functional expression of AAD-1 (v3) in planta for impartingherbicide resistance to 2,4-D and AOPP herbicides is unexpected.

Example 18—Preplant Burndown Applications

This and the following Examples are specific examples of novel herbicideuses made possible by the subject AAD-1 invention.

Preplant burndown herbicide applications are intended to kill weeds thathave emerged over winter or early spring prior to planting a given crop.Typically these applications are applied in no-till or reduced tillagemanagement systems where physical removal of weeds is not completedprior to planting. An herbicide program, therefore, must control a verywide spectrum of broadleaf and grass weeds present at the time ofplanting. Glyphosate, gramoxone, and glufosinate are examples ofnon-selective, non-residual herbicides widely used for preplant burndownherbicide applications. Some weeds, however, are difficult to control atthis time of the season due to one or more of the following: inherentinsensitivity of the weed species or biotype to the herbicide,relatively large size of winter annual weeds, and cool weatherconditions limiting herbicide uptake and activity. Several herbicideoptions are available to tankmix with these herbicides to increasespectrum and activity on weeds where the non-selective herbicides areweak. An example would be 2,4-D tankmix applications with glyphosate toassist in the control of Conyza canadensis (horseweed). Glyphosate canbe used from 420 to 1680 g ae/ha, more typically 560 to 840 g ae/ha, forthe preplant burndown control of most weeds present; however, 280-1120 gae/ha of 2,4-D can be applied to aid in control of many broadleaf weedspecies (e.g., horseweed). 2,4-D is an herbicide of choice because it iseffective on a very wide range of broadleaf weeds, effective even at lowtemperatures, and extremely inexpensive. However, if the subsequent cropis a sensitive dicot crop, 2,4-D residues in the soil (althoughshort-lived) can negatively impact the crop. Soybeans are a sensitivecrop and require a minimum time period of 7 days (for 280 g ae/ha 2,4-Drate) to at least 30 days (for 2,4-D applications of 1120 g ae/ha) tooccur between burndown applications and planting. 2,4-D is prohibited asa burndown treatment prior to cotton planting (see federal labels, mostare available through CPR, 2003 or online at cdms.net/manuf/manuf.asp).With AAD-1 (v3) transformed cotton or soybeans, these crops should beable to survive 2,4-D residues in the soil from burndown applicationsapplied right up to and even after planting before emergence of thecrop. The increased flexibility and reduced cost of tankmix (orcommercial premix) partners will improve weed control options andincrease the robustness of burndown applications in important no-tilland reduced tillage situations. This example is one of many options thatwill be available. Those skilled in the art of weed control will note avariety of other applications including, but not limited togramoxone+2,4-D or glufosinate+2,4-D by utilizing products described infederal herbicide labels (CPR, 2003) and uses described in AgrilianceCrop Protection Guide (2003), as examples. Those skilled in the art willalso recognize that the above example can be applied to any2,4-D-sensitive (or other phenoxy auxin herbicide) crop that would beprotected by the AAD-1 (v3) gene if stably transformed.

Example 19—in-Crop Use of Phenoxy Auxins Herbicides in AAD-1 (v3) OnlyTransformed Soybeans, Cotton, and Other Dicot Crops

AAD-1 (v3) can enable the use of phenoxy auxin herbicides (e.g., 2,4-D,dichlorprop, MCPA, et al.) for the control of a wide spectrum ofbroadleaf weeds directly in crops normally sensitive to 2,4-D.Application of 2,4-D at 280 to 2240 g ae/ha would control most broadleafweed species present in agronomic environments. More typically, 560-1120g ae/ha is used. For a complete weed control system, grass weeds must becontrolled. A variety of broad spectrum graminicide herbicides,including but not limited to haloxyfop, quizalofop, fenoxaprop,fluazifop, sethoxydim, and clethodim are currently registered for use inmost dicot crops which are naturally tolerant to these herbicides. Acombination of quizalofop (20-100 g ae/ha) plus 2,4-D (420-840 g ae/ha)could provide two herbicide modes of action in an AAD-1 (v3)-transformeddicot crop (i.e., soybean or cotton) that would control most agronomicweeds in a similar fashion to glyphosate in glyphosate tolerant crops(see weed control spectra by reference to Agriliance Crop ProtectionGuide performance ratings).

An advantage to this additional tool is the extremely low cost of thebroadleaf herbicide component and potential short-lived residual weedcontrol provided by higher rates of 2,4-D and/or AOPP herbicides whenused at higher rates, whereas a non-residual herbicide like glyphosatewould provide no control of later germinating weeds. This tool alsoprovides a mechanism to rotate herbicide modes of action with theconvenience of HTC as an integrated herbicide resistance and weed shiftmanagement strategy in a glyphosate tolerant crop/AAD-1 (v3) HTCrotation strategy, whether one rotates crops species or not.Additionally, the grass and broadleaf weed control components of thissystem are independent of one another, thus allowing one skilled in theare of weed control to determine the most cost effective and efficaciousratio of auxin and AOPP herbicide. For example, if broadleaf weeds werethe only significant weeds present when herbicide applications wereneeded, an herbicide application of 560 to 1120 g ae/ha 2,4-D could bemade without another herbicide. This would reduce unnecessary herbicideapplications, provide flexibility to reduce input costs and reduceenvironmental loads of pesticides, and reduce unnecessary selectionpressure for the development of herbicide resistant weeds.

Further benefits could include tolerance to 2,4-D drift orvolatilization as mechanisms for off-site 2,4-D injury to dicot crops;no interval required before planting following 2,4-D application (seeprevious example); and fewer problems from contamination injury to dicotcrops resulting from incompletely cleaned bulk tanks that had contained2,4-D. Dicamba (and other herbicides) can still be used for thesubsequent control of AAD-1 (v3)-transformed dicot crop volunteers.

Those skilled in the art will also recognize that the above example canbe applied to any 2,4-D-sensitive (or other phenoxy auxin herbicide)crop that would be protected by the AAD-1 (v3) gene if stablytransformed. One skilled in the art of weed control will now recognizethat use of various commercial phenoxy auxin herbicides alone or incombination with any commercial AOPP herbicide is enabled by AAD-1 (v3)transformation. Specific rates of other herbicides representative ofthese chemistries can be determined by the herbicide labels compiled inthe CPR (Crop Protection Reference) book or similar compilation or anycommercial or academic crop protection references such as the CropProtection Guide from Agriliance (2003). Each alternative herbicideenabled for use in HTCs by AAD-1 (v3), whether used alone, tank mixed,or sequentially, is considered within the scope of this invention.

Example 20—In-Crop Use of Phenoxy Auxins and AOPP Herbicides in AAD-1(v3) Only Transformed Corn, Rice, and Other Monocot Species

In an analogous fashion, transformation of grass species (such as, butnot limited to, corn, rice, wheat, barley, or turf and pasture grasses)with AAD-1 (v3) would allow the use of highly efficacious AOPPgraminicides in crops normally sensitive to these herbicides. Most grassspecies have a natural tolerance to auxinic herbicides such as thephenoxy auxins (i.e., 2,4-D, dichlorprop, et al.). However, a relativelylow level of crop selectivity has resulted in diminished utility inthese crops due to a shortened window of application timing andalternative broadleaf weeds. AAD-1 (v3)-transformed monocot crops would,therefore, enable the use of a similar combination of treatmentsdescribed for dicot crops such as the application of 2,4-D at 280 to2240 g ae/ha to control most broadleaf weed species. More typically,560-1120 g ae/ha would be used. A variety of broad spectrum AOPPgraminicide herbicides (including but not limited to haloxyfop,quizalofop, fenoxaprop, and fluazifop) could be utilized for effectivelycontrolling a wide selection of grass weeds. Cyclohexanedionegraminicidal herbicides like sethoxydim, clethodim, et al. could not beused in this system as shown for dicot crops since AAD-1 would notprotect from this chemistry, and grass crops will be naturally sensitiveto the cyclohexanedione chemistries. However, this attribute wouldenable the use of cyclohexanedione herbicides for the subsequent controlof AAD-1 (v3)-transformed grass crop volunteers. Similar weed controlstrategies are now enabled by AAD-1 for the dicot crop species. Acombination of quizalofop (20-100 g ae/ha) plus 2,4-D (420-840 g ae/ha)could provide two herbicide modes of action in an AAD-1 (v3) transformedmonocot crop (e.g., corn and rice) that would control most agronomicweeds in a similar fashion to glyphosate in glyphosate tolerant crops(see weed control spectra by reference to Agriliance Crop ProtectionGuide performance ratings).

An advantage to this additional tool is the extremely low cost of thebroadleaf herbicide component and potential short-lived residual weedcontrol provided by higher rates of 2,4-D and/or AOPP herbicides whenused at higher rates. In contrast, a non-residual herbicide likeglyphosate would provide no control of later-germinating weeds. Thistool would also provide a mechanism to rotate herbicide modes of actionwith the convenience of HTC as an integrated-herbicide-resistance andweed-shift-management strategy in a glyphosate tolerant crop/AAD-1 (v3)HTC rotation strategy, whether one rotates crops species or not.Additionally, the grass and broadleaf weed control components of thissystem are independent of one another, thus allowing one skilled in theare of weed control to determine the most cost effective and efficaciousratio of auxin and AOPP herbicide. For example, if broadleaf weeds werethe only significant weeds present when herbicide applications wereneeded, an herbicide application of 560 to 1120 g ae/ha 2,4-D could bemade without another herbicide. This would reduce unnecessary herbicideapplications, provide flexibility to reduce input costs and reduceenvironmental loads of pesticides, and reduce unnecessary selectionpressure for the development of herbicide resistant weeds. The increasedtolerance of corn, and other monocots to the phenoxy auxins shall enableuse of these herbicides in-crop without growth stage restrictions or thepotential for crop leaning, unfurling phenomena such as “rat-tailing,”crop leaning, growth regulator-induced stalk brittleness in corn, ordeformed brace roots.

Those skilled in the art will now also recognize that the above examplecan be applied to any monocot crop that would be protected by the AAD-1(v3) gene from injury by any AOPP herbicide. One skilled in the art ofweed control will now recognize that use of various commercial phenoxyauxin herbicides alone or in combination with any commercial AOPPherbicide is enabled by AAD-1 (v3) transformation. Specific rates ofother herbicides representative of these chemistries can be determinedby the herbicide labels compiled in the CPR (Crop Protection Reference)book or similar compilation, labels compiled online (e.g.,cdms.net/manuf/manuf.asp), or any commercial or academic crop protectionguides such as the Crop Protection Guide from Agriliance (2003). Eachalternative herbicide enabled for use in HTCs by AAD-1 (v3), whetherused alone, tank mixed, or sequentially, is considered within the scopeof this invention.

Example 21—AAD-1 (v3) Stacked with Glyphosate Tolerance Trait in anyCrop

The vast majority of cotton, canola, and soybean acres planted in NorthAmerica contain a glyphosate tolerance (GT) trait, and adoption of GTcorn is on the rise. Additional GT crops (e.g., wheat, rice, sugar beet,and turf) have been under development but have not been commerciallyreleased to date. Many other glyphosate resistant species are inexperimental to development stage (e.g., alfalfa, sugar cane, sunflower,beets, peas, carrot, cucumber, lettuce, onion, strawberry, tomato, andtobacco; forestry species like poplar and sweetgum; and horticulturalspecies like marigold, petunia, and begonias;isb.vt.edu/cfdocs/fieldtests1.cfm, 2005 on the World Wide Web). GTC'sare valuable tools for the sheer breadth of weeds controlled andconvenience and cost effectiveness provided by this system. However,glyphosate's utility as a now-standard base treatment is selecting forglyphosate resistant weeds. Furthermore, weeds that glyphosate isinherently less efficacious on are shifting to the predominant speciesin fields where glyphosate-only chemical programs are being practiced.By stacking AAD-1 (v3) with a GT trait, either through conventionalbreeding or jointly as a novel transformation event, weed controlefficacy, flexibility, and ability to manage weed shifts and herbicideresistance development could be improved. As mentioned in previousexamples, by transforming crops with AAD-1 (v3), one can selectivelyapply AOPP herbicides in monocot crops, monocot crops will have a highermargin of phenoxy auxin safety, and phenoxy auxins can be selectivelyapplied in dicot crops. Several scenarios for improved weed controloptions can be envisioned where AAD-1 (v3) and a GT trait are stacked inany monocot or dicot crop species:

-   -   a) Glyphosate can be applied at a standard postemergent        application rate (420 to 2160 g ae/ha, preferably 560 to 840 g        ae/ha) for the control of most grass and broadleaf weed species.        For the control of glyphosate resistant broadleaf weeds like        Conyza canadensis or weeds inherently difficult to control with        glyphosate (e.g., Commelina spp), 280-2240 g ae/ha (preferably        560-1120 g ae/ha) 2,4-D can be applied sequentially, tank mixed,        or as a premix with glyphosate to provide effective control.    -   b) Glyphosate can be applied at a standard postemergent        application rate (420 to 2160 g ae/ha, preferably 560 to 840 g        ae/ha) for the control of most grass and broadleaf weed species.        For the control of glyphosate resistant grass species like        Lolium rigidum or Eleusine indica, 10-200 g ae/ha (preferably        20-100 g ae/ha) quizalofop can be applied sequentially, tank        mixed, or as a premix with glyphosate to provide effective        control.    -   c) Currently, glyphosate rates applied in GTC's generally range        from 560 to 2240 g ae/ha per application timing. Glyphosate is        far more efficacious on grass species than broadleaf weed        species. AAD-1 (v3)+GT stacked traits would allow        grass-effective rates of glyphosate (105-840 g ae/ha, more        preferably 210-420 g ae/ha). 2,4-D (at 280-2240 g ae/ha, more        preferably 560-1120 g ae/ha) could then be applied sequentially,        tank mixed, or as a premix with grass-effective rates of        glyphosate to provide necessary broadleaf weed control. An AOPP        herbicide like quizalofop at 10-200 g ae/ha (preferably 20-100 g        ae/ha and more preferably 20-35 g ae/ha), could be for more        robust grass weed control and/or for delaying the development of        glyphosate resistant grasses. The low rate of glyphosate would        also provide some benefit to the broadleaf weed control;        however, primary control would be from the 2,4-D.

One skilled in the art of weed control will recognize that use of one ormore commercial phenoxy auxin herbicides alone or in combination(sequentially or independently) with one or more commercial AOPPherbicide is enabled by AAD-1 (v3) transformation into crops. Specificrates of other herbicides representative of these chemistries can bedetermined by the herbicide labels compiled in the CPR (Crop ProtectionReference) book or similar compilation, labels compiled online (e.g.,cdms.net/manuf/manuf.asp), or any commercial or academic crop protectionguides such as the Crop Protection Guide from Agriliance (2003). Eachalternative herbicide enabled for use in HTCs by AAD-1 (v3), whetherused alone, tank mixed, or sequentially, is considered within the scopeof this invention.

Example 22—AAD-1 (v3) Stacked with Glufosinate Tolerance Trait in anyCrop

Glufosinate tolerance (PAT or bar) is currently present in a number ofcrops planted in North America either as a selectable marker for aninput trait like insect resistance proteins or specifically as an HTCtrait. Crops include, but are not limited to, glufosinate tolerantcanola, corn, and cotton. Additional glufosinate tolerant crops (e.g.,rice, sugar beet, soybeans, and turf) have been under development buthave not been commercially released to date. Glufosinate, likeglyphosate, is a relatively non-selective, broad spectrum grass andbroadleaf herbicide. Glufosinate's mode of action differs fromglyphosate. It is faster acting, resulting in desiccation and “burning”of treated leaves 24-48 hours after herbicide application. This isadvantageous for the appearance of rapid weed control. However, thisalso limits translocation of glufosinate to meristematic regions oftarget plants resulting in poorer weed control as evidenced by relativeweed control performance ratings of the two compounds in many species(Agriliance, 2003).

By stacking AAD-1 (v3) with a glufosinate tolerance trait, eitherthrough conventional breeding or jointly as a novel transformationevent, weed control efficacy, flexibility, and ability to manage weedshifts and herbicide resistance development could be improved. Asmentioned in previous examples, by transforming crops with AAD-1 (v3),one can selectively apply AOPP herbicides in monocot crops, monocotcrops will have a higher margin of phenoxy auxin safety, and phenoxyauxins can be selectively applied in dicot crops. Several scenarios forimproved weed control options can be envisioned where AAD-1 (v3) and aglufosinate tolerance trait are stacked in any monocot or dicot cropspecies:

-   -   a) Glufosinate can be applied at a standard postemergent        application rate (200 to 1700 g ae/ha, preferably 350 to 500 g        ae/ha) for the control of many grass and broadleaf weed species.        To date, no glufosinate-resistant weeds have been confirmed;        however, glufosinate has a greater number of weeds that are        inherently more tolerant than does glyphosate.        -   i) Inherently tolerant grass weed species (e.g., Echinochloa            spp or Sorghum spp) could be controlled by tank mixing            10-200 g ae/ha (preferably 20-100 g ae/ha) quizalofop.        -   ii) Inherently tolerant broadleaf weed species (e.g.,            Cirsium arvensis and Apocynum cannabinum) could be            controlled by tank mixing 280-2240 g ae/ha, more preferably            560-2240 g ae/ha, 2,4-D for effective control of these more            difficult-to-control perennial species and to improve the            robustness of control on annual broadleaf weed species.    -   b) A three-way combination of glufosinate (200-500 g        ae/ha)+2,4-D (280-1120 g ae/ha)+quizalofop (10-100 g ae/ha), for        example, could provide more robust, overlapping weed control        spectrum. Additionally, the overlapping spectrum provides an        additional mechanism for the management or delay of herbicide        resistant weeds.

One skilled in the art of weed control will recognize that use of one ormore commercial phenoxy auxin herbicides alone or in combination(sequentially or independently) with one or more commercial AOPPherbicide is enabled by AAD-1 (v3) transformation into crops. Specificrates of other herbicides representative of these chemistries can bedetermined by the herbicide labels compiled in the CPR (Crop ProtectionReference) book or similar compilation, labels compiled online (e.g.,cdms.net/manuf/manuf.asp), or any commercial or academic crop protectionguides such as the Crop Protection Guide from Agriliance (2003). Eachalternative herbicide enabled for use in HTCs by AAD-1 (v3), whetherused alone, tank mixed, or sequentially, is considered within the scopeof this invention.

Example 23—AAD-1 (v3) Stacked with AHAS Trait in any Crop

Imidazolinone herbicide tolerance (AHAS, et al.) is currently present ina number of crops planted in North America including, but not limitedto, corn, rice, and wheat. Additional imidazolinone tolerant crops(e.g., cotton and sugar beet) have been under development but have notbeen commercially released to date. Many imidazolinone herbicides (e.g.,imazamox, imazethapyr, imazaquin, and imazapic) are currently usedselectively in various conventional crops. The use of imazethapyr,imazamox, and the non-selective imazapyr has been enabled throughimidazolinone tolerance traits like AHAS et al. Imidazolinone tolerantHTCs to date have the advantage of being non-transgenic. This chemistryclass also has significant soil residual activity, thus being able toprovide weed control extended beyond the application timing, unlikeglyphosate or glufosinate-based systems. However, the spectrum of weedscontrolled by imidazolinone herbicides is not as broad as glyphosate(Agriliance, 2003). Additionally, imidazolinone herbicides have a modeof action (inhibition of acetolactate synthase, ALS) to which many weedshave developed resistance (Heap, 2004). By stacking AAD-1 (v3) with animidazolinone tolerance trait, either through conventional breeding orjointly as a novel transformation event, weed control efficacy,flexibility, and ability to manage weed shifts and herbicide resistancedevelopment could be improved. As mentioned in previous examples, bytransforming crops with AAD-1 (v3), one can selectively apply AOPPherbicides in monocot crops, monocot crops will have a higher margin ofphenoxy auxin safety, and phenoxy auxins can be selectively applied indicot crops. Several scenarios for improved weed control options can beenvisioned where AAD-1 (v3) and an imidazolinone tolerance trait arestacked in any monocot or dicot crop species:

-   -   a) Imazethapyr can be applied at a standard postemergent        application rate of (35 to 280 g ae/ha, preferably 70-140 g        ae/ha) for the control of many grass and broadleaf weed species.        -   i) ALS-inhibitor resistant broadleaf weeds like Amaranthus            rudis, Ambrosia trifida, Chenopodium album (among others,            Heap, 2004) could be controlled by tank mixing 280-2240 g            ae/ha, more preferably 560-1120 g ae/ha, 2,4-D.        -   ii) Inherently more tolerant broadleaf species to            imidazolinone herbicides like Ipomoea spp. can also be            controlled by tank mixing 280-2240 g ae/ha, more preferably            560-1120 g ae/ha, 2,4-D.        -   iii) ALS-inhibitor resistant grass weeds like Sorghum            halepense and Lolium spp. can be controlled by tank mixing            10-200 g ae/ha (preferably 20-100 g ae/ha) quizalofop.        -   iv) Inherently tolerant grass weed species (e.g., Agropyron            repens) could also be controlled by tank mixing 10-200 g            ae/ha (preferably 20-100 g ae/ha) quizalofop.    -   b) A three-way combination of imazethapyr (35 to 280 g ae/ha,        preferably 70-140 g ae/ha)+2,4-D (280-1120 g ae/ha)+quizalofop        (10-100 g ae/ha), for example, could provide more robust,        overlapping weed control spectrum. Additionally, the overlapping        spectrum provides an additional mechanism for the management or        delay of herbicide resistant weeds.

One skilled in the art of weed control will recognize that use of any ofvarious commercial imidazolinone herbicides, phenoxy auxin herbicides,or AOPP herbicide, alone or in multiple combinations, is enabled byAAD-1 (v3) transformation and stacking with any imidazolinone tolerancetrait either by conventional breeding or genetic engineering. Specificrates of other herbicides representative of these chemistries can bedetermined by the herbicide labels compiled in the CPR (Crop ProtectionReference) book or similar compilation, labels compiled online (e.g.,cdms.net/manuf/manuf.asp), or any commercial or academic crop protectionguides such as the Crop Protection Guide from Agriliance (2003). Eachalternative herbicide enabled for use in HTCs by AAD-1 (v3), whetherused alone, tank mixed, or sequentially, is considered within the scopeof this invention.

Example 24—AAD-1 (v3) in Rice

24.1—Media Description.

Culture media employed were adjusted to pH 5.8 with 1 M KOH andsolidified with 2.5 g/l Phytagel (Sigma). Embryogenic calli werecultured in 100×20 mm Petri dishes containing 40 ml semi-solid medium.Rice plantlets were grown on 50 ml medium in Magenta boxes. Cellsuspensions were maintained in 125-ml conical flasks containing 35 mlliquid medium and rotated at 125 rpm. Induction and maintenance ofembryogenic cultures took place in the dark at 25-26° C., and plantregeneration and whole-plant culture took place in a 16-h photoperiod(Zhang et al. 1996).

Induction and maintenance of embryogenic callus took place on NB basalmedium as described previously (Li et al. 1993), but adapted to contain500 mg/l glutamine Suspension cultures were initiated and maintained inSZ liquid medium (Zhang et al. 1998) with the inclusion of 30 g/lsucrose in place of maltose. Osmotic medium (NBO) consisted of NB mediumwith the addition of 0.256 M each of mannitol and sorbitol.Hygromycin-B-resistant callus was selected on NB medium supplementedwith 50 mg/l hygromycin B for 3-4 weeks. Pre-regeneration took place onmedium (PRH50) consisting of NB medium without 2,4-dichlorophenoxyaceticacid (2,4-D), but with the addition of 2 mg/l 6-benzylaminopurine (BAP),1 mg/l α-naphthaleneacetic acid (NAA), 5 mg/l abscisic acid (ABA) and 50mg/l hygromycin B for 1 week. Regeneration of plantlets followed viaculture on regeneration medium (RNH50) comprising NB medium without2,4-D, and supplemented with 3 mg/l BAP, 0.5 mg/l NAA, and 50 mg/lhygromycin B until shoots regenerated. Shoots were transferred torooting medium with half-strength Murashige and Skoog basal salts and

Gamborg's B5 vitamins, supplemented with 1% sucrose and 50 mg/lhygromycin B (½MSH50).

24.2—Tissue Culture Development.

Mature desiccated seeds of Oryza sativa L. japonica cv. Taipei 309 weresterilized as described in Zhang et al. 1996. Embryogenic tissues wereinduced by culturing sterile mature rice seeds on NB medium in the dark.The primary callus approximately 1 mm in diameter, was removed from thescutellum and used to initiate cell suspension in SZ liquid medium.Suspensions were then maintained as described in Zhang 1995.Suspension-derived embryogenic tissues were removed from liquid culture3-5 days after the previous subculture and placed on NBO osmotic mediumto form a circle about 2.5 cm across in a Petri dish and cultured for 4h prior to bombardment. Sixteen to 20 h after bombardment, tissues weretransferred from NBO medium onto NBH50 hygromycin B selection medium,ensuring that the bombarded surface was facing upward, and incubated inthe dark for 14-17 days. Newly formed callus was then separated from theoriginal bombarded explants and placed nearby on the same medium.Following an additional 8-12 days, relatively compact, opaque callus wasvisually identified, and transferred to PRH50 pre-regeneration mediumfor 7 days in the dark. Growing callus, which became more compact andopaque was then subcultured onto RNH50 regeneration medium for a periodof 14-21 days under a 16-h photoperiod. Regenerating shoots weretransferred to Magenta boxes containing ½ MSH50 medium. Multiple plantsregenerated from a single explant are considered siblings and weretreated as one independent plant line. A plant was scored as positivefor the hph gene if it produced thick, white roots and grew vigorouslyon ½ MSH50 medium. Once plantlets had reached the top of Magenta boxes,they were transferred to soil in a 6-cm pot under 100% humidity for aweek, then moved to a growth chamber with a 14-h light period at 30° C.and in the dark at 21° C. for 2-3 weeks before transplanting into 13-cmpots in the greenhouse. Seeds were collected and dried at 37° C. for oneweek prior to storage at 4° C.

24.3—Microprojectile Bombardment.

All bombardments were conducted with the Biolistic PDS-1000/He™ system(Bio-Rad, Laboratories, Inc.). Three milligrams of 1.0 micron diametergold particles were washed once with 100% ethanol, twice with steriledistilled water and resuspended in 50 μl water in a siliconizedEppendorf tube. Five micrograms plasmid DNA representing a 1:6 molarratio of pDOW3303 (Hpt-containing vector) to pDAB3403, 20 μl spermidine(0.1 M) and 50 calcium chloride (2.5 M) were added to the goldsuspension. The mixture was incubated at room temperature for 10 min,pelleted at 10000 rpm for 10 s, resuspended in 60 μl cold 100% ethanoland 8-9 μl was distributed onto each macrocarrier. Tissue samples werebombarded at 1100 psi and 27 in of Hg vacuum as described by Zhang etal. (1996).

24.4—Tolerance Testing.

Rice plantlets at the 3-5 leaf stage were sprayed with a 0.3% (v/v)solution of DuPont™ Assure® II containing 1% (v/v) Agridex crop oilconcentrate using a DeVilbiss bulb sprayer (model 15-RD glass atomizer).This concentration corresponds to approximately 140 g ae/ha. Each plantwas spayed in a fume hood at a distance of 8-12 inches with 6 fullsquirts of the sprayer directed so that the entire plant was coveredwith an equal portion of herbicide. Each squirt delivered approximately100 μl solution to the plantlet. Once sprayed, plantlets were allowed todry for one hour before being moved out of the fume hood. Rating forsensitivity or resistance was done at 10-14 days after treatment (DAT)and is shown in Table 35 below.

TABLE 35 Sample Name 140 g ae/ha quizalofop Control Dead 63-1A No injury63-1F No injury 63-4B No injury 63-4D No injury 63-6C Dead

24.5—Tissue Harvesting, DNA Isolation and Quantification.

Fresh tissue was placed into tubes and lyophilized at 4° C. for 2 days.After the tissue was fully dried, a tungsten bead (Valenite) was placedin the tube and the samples were subjected to 1 minute of dry grindingusing a Kelco bead mill. The standard DNeasy DNA isolation procedure wasthen followed (Qiagen, Dneasy 69109). An aliquot of the extracted DNAwas then stained with Pico Green (Molecular Probes P7589) and scanned inthe florometer (BioTek) with known standards to obtain the concentrationin ng/μ1.

24.6—Southern Blot Analysis.

Southern blot analysis was performed with total DNA obtained from theQiagen DNeasy kit. A total of 2 μg of DNA was subjected to an overnightdigest of HindIII for pDAB3403 to obtain integration data. Likewise atotal of 2 ug of DNA was subjected to an overnight digest of MfeI toobtain the PTU data. After the overnight digestion an aliquot of ˜100 ngwas run on a 1% gel to ensure complete digestion. After this assurancethe samples were run on a large 0.85% agarose gel overnight at 40 volts.The gel was then denatured in 0.2 M NaOH, 0.6 M NaCl for 30 minutes. Thegel was then neutralized in 0.5 M Tris HCl, 1.5 M NaCl pH of 7.5 for 30minutes. A gel apparatus containing 20×SSC was then set-up to obtain agravity gel to nylon membrane (Millipore INYC00010) transfer overnight.After the overnight transfer the membrane was then subjected to UV lightvia a crosslinker (Stratagene UV stratalinker 1800) at 1200 X100microjoules. The membrane was then washed in 0.1% SDS, 0.1 SSC for 45minutes. After the 45 minute wash, the membrane was baked for 3 hours at80° C. and then stored at 4° C. until hybridization. The hybridizationtemplate fragment was prepared using coding region PCR using plasmidpDAB3404. A total of 100 ng of total DNA was used as template. 20 mM ofeach primer was used with the Takara Ex Taq PCR Polymerase kit (MirusTAKRROO1A). Primers for Southern fragment PCR AAD-1 were(Forward—ATGGCTCATGCTGCCCTCAGCC) (SEQ ID NO:31) and(Reverse—GGGCAGGCCTAACTCCACCAA) (SEQ ID NO:32). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 65° C. for 30 seconds, and 72° C. for 1 minute and 45seconds followed by 72° C. for 10 minutes.

The product was run on a 1% agarose gel and excised then gel extractedusing the Qiagen (28706) gel extraction procedure. The membrane was thensubjected to a pre-hybridization at 60° C. step for 1 hour in PerfectHyb buffer (Sigma H7033). The Prime it RmT dCTP-labeling reaction(Stratagene 300392) procedure was used to develop the p32 based probe(Perkin Elmer). The probe was cleaned-up using the Probe Quant. G50columns (Amersham 27-5335-01). Two million counts CPM were used tohybridize the Southern blots overnight. After the overnighthybridization the blots were then subjected to two 20 minute washes at65° C. in 0.1% SDS, 0.1 SSC. The blots were then exposed to a phosphorimage screen overnight and scanned using a storm scanner (MOLECULARDEVICES). A summary of the results is presented in Table 36.

TABLE 36 Southern Results. Integration Southern data PTU Southern dataEvent Number of bands Expected size 3049 bp 63-1 A 8 yes, 7 distinctbands 63-1 F 5 yes, 9 distinct bands 63-4 A 20 yes, 20 distinct bands63-4 D 20 yes, 19 distinct bands 63-6 C 2 Insufficient DNA yield forboth cuts

Plants 63-1 A and 63-1 F are not the same event; Plants 63-4 A and 63-4D are the same event. These events have the expected size PTU's, butthey are very complex. This Southern blot PTU data correlates withexpression data and the spray data. Sample 63-6 C did not have enoughDNA present to perform both Integration and PTU southern blots.

24.7—Western Data

Sample preparation and analysis conditions were as described previously.Five transgenic rice lines and 1 non-transgenic control were analyzedfor AAD-1 expression using ELISA and Western blot. AAD-1 was detected infour lines (63-1A, 63-1F, 63-4B and 63-4D) but not in line 63-1C or thecontrol plant. Expression levels ranged from 15.6 to 183 ppm of totalsoluble protein. A summary of the results is presented in Table 37.

TABLE 37 ELISA Sample TSP [AAD1] Expression Lane Name (μg/mL) (ng/mL)(ppm) Western 1 Control 6719.58 0.00 0.00 − 2 63-1A 8311.87 351.17 42.25± 3 63-1F 11453.31 2092.35 182.69 ++ 4 63-4B 13835.09 216.00 15.61 + 536-4D 13656.49 717.05 52.51 ++ 6 63-6C 5343.63 0.00 0.00 − 7 AAD1Standard (0.5 μg/mL) +++ 8 AAD1 Standard (5.0 μg/mL) +++++

Example 25—Turf Grass Transformation Procedures

Genetic transformation, with AAD-1 (v3) substituted for the “bar” geneas described below, of creeping bentgrass mediated by Agrobacteriumtumefaciens could be achieved through embryogenic callus initiated fromseeds (cv. Penn-A-4), as described generally below. See “Efficiency ofAgrobacterium tumefaciens-mediated turfgrass (Agrostis stolonifera L)transformation” (Luo et. al., 2004).

Callus is infected with an A. tumefaciens strain (LBA4404) harboring asuper-binary vector that contained an herbicide-resistant bar genedriven either by a rice ubiquitin promoter. The overall stabletransformation efficiency ranged from 18% to 45%. Southern blot andgenetic analysis confirmed transgene integration in the creepingbentgrass genome and normal transmission and stable expression of thetransgene in the T₁ generation. All independent transformation eventscarried one to three copies of the transgene, and a majority (60-65%)contained only a single copy of the foreign gene with no apparentrearrangements.

25.1—Seed Preparation for Embryogenic Callus Induction.

Mature seeds were dehusked with sand paper and surface sterilized in 10%(v/v) Clorox bleach (6% sodium hypochlorite) plus 0.2% (v/v) Tween 20(Polysorbate 20) with vigorous shaking for 90 min Following rinsing fivetimes in sterile distilled water, the seeds were placed ontocallus-induction medium (MS basal salts and vitamins, 30 g/l sucrose,500 mg/l casein hydrolysate, 6.6 mg/l 3,6-dichloro-o-anisic acid(dicamba), 0.5 mg/l 6-benzylaminopurine (BAP) and 2 g/l Phytagel. The pHof the medium was adjusted to 5.7 before autoclaving at 120° C. for 20min).

25.2—Embryogenic Callus Induction.

The culture plates containing prepared seed explants were kept in thedark at room temperature for 6 weeks. Embryogenic calli were visuallyselected and subcultured on fresh callus-induction medium in the dark atroom temperature for 1 week before co-cultivation.

25.3—Agrobacterium Infection and Co-Cultivation.

One day before agro-infection, the embryogenic callus was divided into1- to 2-mm pieces and placed on callus-induction medium containing 100μM acetosyringone. A 10-ul aliquot of Agrobacterium (LBA4404) suspension(0D=1.0 at 660 nm) was then applied to each piece of callus, followed by3 days of co-cultivation in the dark at 25° C.

25.4—Resting Stage and Agrobacterium Control.

For the antibiotic treatment step, the callus was then transferred andcultured for 2 weeks on callus-induction medium plus 125 mg/l cefotaximeand 250 mg/l carbenicillin to suppress bacterial growth.

25.5—Selection and Potential Transgenic Colony Identification.

Subsequently, for selection, the callus was moved to callus-inductionmedium containing 250 mg/l cefotaxime and 10 mg/l phosphinothricin (PPT)for 8 weeks. Antibiotic treatment and the entire selection process wereperformed at room temperature in the dark. The subculture intervalduring selection was typically 3 weeks.

25.6—Regeneration of Transgenic Plants.

For plant regeneration, the PPT-resistant proliferating callus eventsare first moved to regeneration medium (MS basal medium, 30 g/l sucrose,100 mg/l myo-inositol, 1 mg/l BAP and 2 g/l Phytagel) supplemented withcefotaxime, PPT or hygromycin. These calli were kept in the dark at roomtemperature for 1 week and then moved into the light for 2-3 weeks todevelop shoots. No Albino plants with PPT selection (Hygromycin if usedas a selection agent, produces a high level of albino plants).

25.7—Root Induction and Transfer to Greenhouse.

Small shoots were then separated and transferred to hormone-freeregeneration medium containing PPT and cefotaxime to promote root growthwhile maintaining selection pressure and suppressing any remainingAgrobacterium cells. Plantlets with well-developed roots (3-5 weeks)were then transferred to soil and grown either in the greenhouse or inthe field.

25.8—Vernalization and Out-Crossing of Transgenic Plants.

Transgenic plants were maintained out of doors in a containment nursery(3-6 months) until the winter solstice in December. The vernalizedplants were then transferred to the greenhouse and kept at 25° C. undera 16/8 h photoperiod and surrounded by non-transgenic wild-type plantsthat physically isolated them from other pollen sources. The plantsstarted flowering 3-4 weeks after being moved back into the greenhouse.They were out-crossed with the pollen from the surrounding wild typeplants. The seeds collected from each individual transgenic plant weregerminated in soil at 25° C., and T₁ plants were grown in the greenhousefor further analysis.

25.9—Other Target Grasses.

Other grasses that can be targeted for AAD-1 transformation according tothe subject invention include Annual meadowgrass (Poa annua),Bahiagrass, Bentgrass, Bermudagrass, Bluegrass, Bluestems, Bromegrass,Browntop bent (Agrostis capillaries), Buffalograss, Canary Grass,Carpetgrass, Centipedegrass, Chewings fescue (Festuca rubra commutate),Crabgrass, Creeping bent (Agrostis stolonifera), Crested hairgrass(Koeleria macrantha), Dallisgrass, Fescue, Festolium, Hard/sheeps fescue(Festuca ovina), Gramagrass, Indiangrass, Johnsongrass, Lovegrass, mixes(Equine, Pasture, etc.), Native Grasses, Orchardgrass, Perennialryegrass (Lolium perenne), Redtop, Rescuegrass, annual and perennialRyegrass, Slender creeping red fescue (Festuca rubra trichophylla),Smooth-stalked meadowgrass (Poa pratensis), St. Augustine, Strongcreeping red fescue (Festuca rubra rubra), Sudangrass, Switchgrass, Tallfescue (Festuca arundinacea), Timothy, Tufted hairgrass (Deschampsiacaespitosa), Turfgrasses, Wheatgrass, and Zoysiagrass.

Example 26—AAD-1 (v3) in Canola

26.1—Canola Transformation.

The AAD-1 (v3) gene conferring resistance to 2,4-D was used to transformBrassica napus var. Nexera* 710 with Agrobacterium-mediatedtransformation. The construct contained AAD-1 (v3) gene driven by CsVMVpromoter and Pat gene driven by AtUbi10 promoter.

Seeds were surface-sterilized with 10% commercial bleach for 10 minutesand rinsed 3 times with sterile distilled water. The seeds were thenplaced on one half concentration of MS basal medium (Murashige andSkoog, 1962) and maintained under growth regime set at 25° C., and aphotoperiod of 16 hrs light/8 hrs dark.

Hypocotyl segments (3-5 mm) were excised from 5-7 day old seedlings andplaced on callus induction medium K1D1 (MS medium with 1 mg/l kinetinand 1 mg/l 2,4-D) for 3 days as pre-treatment. The segments were thentransferred into a petri plate, treated with Agrobacterium Z7075 orLBA4404 strain containing pDAB721. The Agrobacterium was grown overnightat 28° C. in the dark on a shaker at 150 rpm and subsequentlyre-suspended in the culture medium.

After 30 min treatment of the hypocotyl segments with Agrobacterium,these were placed back on the callus induction medium for 3 days.Following co-cultivation, the segments were placed K1D1TC (callusinduction medium containing 250 mg/l Carbenicillin and 300 mg/lTimentin) for one week of recovery. Alternately, the segments wereplaced directly on selection medium K1D1H1 (above medium with 1 mg/lHerbiace). Carbenicillin and Timentin were the antibiotics used to killthe Agrobacterium. The selection agent Herbiace allowed the growth ofthe transformed cells.

Callus samples from 35 independent events were tested by PCR. All the 35samples tested positive for the presence of AAD-1 (v3), whereas thenon-transformed controls were negative (section on PCR assay). Tencallus samples were confirmed to express the AAD-1 protein as determinedby ELISA (section on protein analysis).

Callused hypocotyl segments were then placed on B3Z1H1 (MS medium, 3mg/l benzylamino purine, 1 mg/l Zeatin, 0.5 gm/l MES [2-(N-morpholino)ethane sulfonic acid], 5 mg/l silver nitrate, 1 mg/l Herbiace,Carbenicillin and Timentin) shoot regeneration medium. After 3 weeksshoots started regenerating. Hypocotyl segments along with the shootsare transferred to B3Z1H3 medium (MS medium, 3 mg/l benzylamino purine,1 mg/l Zeatin, 0.5 gm/l MES [2-(N-morpholino) ethane sulfonic acid], 5mg/l silver nitrate, 3 mg/l Herbiace, Carbenicillin and Timentin) foranother 3 weeks.

Shoots were excised from the hypocotyl segments and transferred to shootelongation medium MESH10 (MS, 0.5 gm/l MES, 10 mg/l Herbiace,Carbenicillin, Timentin) for 2-4 weeks. The elongated shoots arecultured for root induction on MSI.1 (MS with 0.1 mg/l Indolebutyricacid). Once the plants had a well established root system, these weretransplanted into soil. The plants were acclimated under controlledenvironmental conditions in the Conviron for 1-2 weeks before transferto the greenhouse.

The transformed T0 plants were self-pollinated in the greenhouse toobtain T1 seed. The T0 plants and T1 progeny were sprayed with a rangeof herbicide concentrations to establish the level of protection by theAAD-1 (v3) gene.

26.2—“Molecular Analysis”: Canola Materials and Methods

26.2.1—Tissue harvesting DNA isolation and quantification. Fresh tissuewas placed into tubes and lyophilized at 4° C. for 2 days. After thetissue was fully dried, a tungsten bead (Valenite) was placed in thetube and the samples were subjected to 1 minute of dry grinding using aKelco bead mill. The standard DNeasy DNA isolation procedure was thenfollowed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA wasthen stained with Pico Green (Molecular Probes P7589) and read in theflorometer (BioTek) with known standards to obtain the concentration inng/ul.

26.2.2—Polymerase chain reaction. A total of 100 ng of total DNA wasused as the template. 20 mM of each primer was used with the Takara ExTaq PCR Polymerase kit (Mirus TAKRROO1A). Primers for Coding Region PCRAAD-1 (v3) were (Forward—ATGGCTCATG CTGCCCTCAGCC) (SEQ ID NO:27) and(Reverse—CGGGCAGGCCTAACTCCACCAA) (SEQ ID NO:28). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 65° C. for 30 seconds, and 72° C. for 2 minutes followedby 72° C. for 10 minutes. PCR products were analyzed by electrophoresison a 1% agarose gel stained with EtBr. 35 samples from 35 plants withAAD-1 (v3) events tested positive. Three negative control samples testednegative.

26.3—ELISA.

Using established ELISA described in previous section, AAD-1 protein wasdetected in 10 different canola transformation events. Expression levelsranged from 150 to over 1000 ppm of total soluble protein (TSP). Threedifferent untransformed calli samples were tested in parallel withlittle signal detected, indicating that the antibodies used in the assayhave minimal cross reactivity to the canola cell matrix. A summary ofthe results is presented in Table 38.

TABLE 38 Expression of AAD1 in Canola calli. Weight [TSP] [AAD1]Expression PCR for Sample # (mg) (μg/mL) (ng/mL) (ppm TSP) AAD1 1 114757.02 119.36 157.67 + 2 55 839.79 131.84 156.99 + 3 53 724.41 202.12279.01 + 4 52 629.01 284.89 452.92 + 5 55 521.75 175.88 337.08 + 6 61707.69 74.24 153.71 + 7 51 642.02 559.11 1026.73 + 8 65 707.69 270.73382.56 + 9 51 642.02 197.90 308.25 + 10 51 1417.42 220.63 156.66 +Control 1 53 2424.67 18.67 7.70 − Control 2 61 2549.60 35.00 13.73 −Control 3 59 2374.41 22.79 9.60 −

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1. A transgenic maize plant cell comprising a recombinant polynucleotidethat encodes an AAD-1 protein that exhibits aryloxyalkanoate dioxygenaseactivity wherein said activity enzymatically degrades a phenoxy auxinherbicide and an (R)-aryloxyphenoxypropionate herbicide, further whereinsaid AAD-1 protein comprises: i) an amino acid sequence having at least85% sequence identity with SEQ ID NO: 9; and ii) an AAD-1 motif havingthe general formula of: (SEQ ID NO: 34)HX₁₁₂D(X)₁₁₄₋₁₃₇T(X)₁₃₉₋₂₆₉H(X)₂₇₁₋₂₈₀R,

wherein X₁₁₂ represents a single amino acid at position 112, relative tothe sequence of SEQ ID NO: 9; (X)₁₁₄₋₁₃₇ represents a sequence of 24amino acids; (X)₁₃₉₋₂₆₉ represents a sequence of 131 amino acids; and(X)₂₇₁₋₂₈₀ represents a sequence of 10 amino acids.
 2. The plant cell ofclaim 1 wherein said AAD-1 motif has the general formula of:(SEQ ID NO: 35) HX₁₁₂D(X)₁₁₄₋₁₃₇T(X)₁₃₉₋₂₆₉H(X)₂₇₁₋₂₈₀R(X)₂₈₂₋₂₈₄R,

wherein (X)₂₈₂₋₂₈₄ represents a sequence of 3 amino acids.
 3. Atransgenic plant comprising a plurality of the maize plant cell of claim1, wherein expression of said polynucleotide renders said plant tolerantto an aryloxyalkanoate herbicide.
 4. The plant of claim 3 wherein saidaryloxyalkanoate herbicide is a phenoxy auxin herbicide.
 5. The plant ofclaim 3 wherein said aryloxyalkanoate herbicide is selected from thegroup consisting of 2,4-dichlorophenoxyacetic acid, MCPA, dichlorprop,and mecoprop.
 6. The plant of claim 3 wherein said aryloxyalkanoateherbicide is an (R)-aryloxyphenoxypropionate.
 7. The plant of claim 3wherein said aryloxyalkanoate herbicide is selected from the groupconsisting of (R)-fluazifop, (R)-haloxyfop, (R)-diclofop,(R)-quizalofop, (R)-fenoxaprop, (R)-metamifop, (R)-cyhalofop, and(R)-clodinofop.
 8. The plant of claim 3 wherein said plant furthercomprises a second herbicide resistance gene.
 9. The plant of claim 8wherein said second herbicide resistance gene renders said plantresistant to an herbicide selected from the group consisting ofglyphosate, glufosinate, acetolactate synthase (ALS) inhibitors,inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), dicamba, andinhibitors of protoporphyrinogen oxidase (PPO).
 10. A method ofcontrolling at least one weed in a field, wherein said field contains atleast one plant of claim 3, wherein said method comprises applying to atleast a portion of said field a first herbicide selected from the groupconsisting of a phenoxy auxin herbicide and an(R)-aryloxyphenoxypropionate herbicide.
 11. The method of claim 10wherein said phenoxy auxin herbicide is an R-enantiomer of a chiralphenoxy auxin.
 12. The method of claim 10 wherein said phenoxy auxinherbicide is an achiral phenoxy auxin selected from the group consistingof 2,4-D and MCPA.
 13. The method of claim 10 wherein said(R)-aryloxyphenoxypropionate herbicide is selected from the groupconsisting of (R)-fluazifop, (R)-haloxyfop, (R)-diclofop,(R)-quizalofop, (R)-fenoxaprop, (R)-metamifop, (R)-cyhalofop, and(R)-clodinofop.
 14. The method of claim 10 wherein said method comprisesapplying a second herbicide.
 15. The method of claim 14 wherein saidfirst herbicide and said second herbicide are applied sequentially. 16.The method of claim 14 wherein said first herbicide and said secondherbicide are applied concurrently.
 17. The method of claim 14 whereinsaid first herbicide is a phenoxy auxin and said second herbicide is an(R)-aryloxyphenoxypropionate.
 18. The method of claim 14 wherein saidsecond herbicide is selected from the group consisting of glyphosate,glufosinate, dicamba, acetolactate synthase (ALS) inhibitors,protoporphyrinogen oxidase (PPO) inhibitors, and4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) inhibitors.
 19. The methodof claim 14, wherein said first herbicide is 2,4-D and said secondherbicide is glyphosate or glufosinate.
 20. The method of claim 14,wherein said first herbicide is an (R)-aryloxyphenoxypropionate and saidsecond herbicide is glyphosate or glufosinate.
 21. The method of claim14 wherein said plant further comprises a second herbicide resistancegene that renders said plant resistant to said second herbicide.
 22. Themethod of claim 21 wherein said second gene is selected from the groupconsisting of a modified acetolactate synthase (ALS) gene, a glyphosateresistance gene, a glufosinate resistance gene, and a dicamba-degradingenzyme gene.
 23. The method of claim 14 wherein said method furthercomprises applying a third herbicide.
 24. The method of claim 23,wherein said third herbicide is selected from the group consisting ofglyphosate, glufosinate, HPPD-inhibitors, PPO-inhibitors, ALSinhibitors, and dicamba.
 25. The method of claim 24 wherein said first,second and third herbicides are 2,4-D, quizalofop, and glyphosate.
 26. Aseed comprising a plurality of the plant cell of claim
 1. 27. A methodof controlling weeds in a field, wherein said method comprises applyingan aryloxyalkanoate herbicide to said field and planting a seed of claim26 in said field within 14 days of applying said aryloxyalkanoateherbicide.
 28. A plant grown from the seed of claim
 26. 29. A part,progeny, or asexual propagate of the plant of claim 28, wherein saidpart, progeny, or sexual propagate comprises said polynucleotide.
 30. An(R)-aryloxyphenoxypropionate herbicide tolerant transgenic maize plantcell comprising a recombinant polynucleotide that encodes an AAD-1protein that exhibits aryloxyalkanoate dioxygenase activity wherein saidactivity enzymatically degrades a phenoxy auxin herbicide and an(R)-aryloxyphenoxypropionate herbicide, further wherein said AAD-1protein comprises: i) an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 9; and ii) an AAD-1 motif having thegeneral formula of: (SEQ ID NO: 34)HX₁₁₂D(X)₁₁₄₋₁₃₇T(X)₁₃₉₋₂₆₉H(X)₂₇₁₋₂₈₀R,

wherein X₁₁₂ represents a single amino acid at position 112, relative tothe sequence of SEQ ID NO: 9; (X)₁₁₄₋₁₃₇ represents a sequence of 24amino acids; (X)₁₃₉₋₂₆₉ represents a sequence of 131 amino acids; and(X)₂₇₁₋₂₈₀ represents a sequence of 10 amino acids, wherein said motifhas 90% sequence identity with corresponding amino acids of position 111to 281 of SEQ ID NO: 9.